CHARACTERIZATION OF THE DISCRIMINATIVE STIMULUS EFFECTS OF LORCASERIN IN RATS

PubMed Central

Serafine, Katherine M.; Rice, Kenner C.; France, Charles P.

2016-01-01

Lorcaserin is approved by the Food and Drug Administration for treating obesity and is under consideration for treating substance use disorders; it has agonist properties at serotonin (5-HT)2C receptors and might also have agonist properties at other 5-HT receptor subtypes. This study used drug discrimination to investigate the mechanism(s) of action of lorcaserin. Male Sprague-Dawley rats discriminated 0.56 mg/kg i.p. lorcaserin from saline while responding under a fixed-ratio 5 schedule for food. Lorcaserin (0.178–1.0 mg/kg) dose-dependently increased lorcaserin-lever responding. The 5-HT2C receptor agonist mCPP and the 5-HT2A receptor agonist DOM each occasioned greater than 90% lorcaserin-lever responding in seven of eight rats. The 5-HT1A receptor agonist 8-OH-DPAT occasioned greater than 90% lorcaserin-lever responding in four of seven rats. The 5-HT2C receptor selective antagonist SB 242084 attenuated lorcaserin-lever responding in all eight rats and the 5-HT2A receptor selective antagonist MDL 100907 attenuated lorcaserin-lever responding in six of seven rats. These results suggest that, in addition to agonist properties at 5-HT2C receptors, lorcaserin also has agonist properties at 5-HT2A and 5-HT1A receptors. Because some drugs with 5-HT2A receptor agonist properties are abused, it is important to fully understand the behavioral effects of lorcaserin while considering its potential for treating substance use disorders. PMID:27640338

Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

PubMed

Schindler, Charles W; Karcz-Kubicha, Marzena; Thorndike, Eric B; Müller, Christa E; Tella, Srihari R; Ferré, Sergi; Goldberg, Steven R

2005-03-01

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery.

Assessments of cellular melatonin receptor signaling pathways: β-arrestin recruitment, receptor internalization, and impedance variations.

PubMed

Dupré, Clémence; Bruno, Olivier; Bonnaud, Anne; Giganti, Adeline; Nosjean, Olivier; Legros, Céline; Boutin, Jean A

2018-01-05

Melatonin receptors belong to the family of G-protein coupled receptors. Agonist-induced receptor activation is terminated with the recruitment of β-arrestin, which leads to receptor internalization. Furthermore, agonist binding induces a shift in cellular shape that translates into a change in the electric impedance of the cell. In the present study, we employed engineered cells to study these internalization-related processes in the context of the two melatonin receptors, MT 1 and MT 2 . To assess these three receptor internalization-related functions and validate the results, we employed four classical ligands of melatonin receptors: the natural agonist melatonin; the super-agonist 2-iodo-melatonin and the two antagonists luzindole and 4-phenyl-2-propionamidotetralin. The assessments confirmed the nature of the agonistic ligands but showed that 4-phenyl-2-propionamidotetralin, a described antagonist, is a biased partial agonist at MT 2 with poorer affinity for MT 1 . The methods are now available to be applied to any receptor system for which multiple signaling pathways must be evaluated for new molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

PubMed Central

Mortensen, Martin; Kristiansen, Uffe; Ebert, Bjarke; Frølund, Bente; Krogsgaard-Larsen, Povl; Smart, Trevor G

2004-01-01

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E ∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel. PMID:14990676

Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors

PubMed Central

Huang, Xi-Ping; Nagy, Peter I; Williams, Frederick E; Peseckis, Steven M; Messer, William S

1999-01-01

Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala.HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors.The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors.Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor. PMID:10188986

[Effect of (+/-)-pindolol on the central 5-HT1A receptor by the use of in vivo microdialysis and hippocampal slice preparations].

PubMed

Tsuji, Keiichiro

2002-06-01

Although it is suggested that (+/-)-pindolol, a beta-adrenergic/5-HT1A receptor antagonist, may enhance the efficacy of selective serotonin reuptake inhibitors (SSRI), the results of double-blind studies are contradictory and recent animal studies suggest that (+/-)-pindolol may act as a partial agonist to the 5-HT1A receptor. In this study we have investigated the effect of (+/-)-pindolol on both pre- and postsynaptic 5-HT1A receptors using in vivo microdialysis and hippocampal slice preparations. (+/-)-pindolol and flesinoxan, a 5-HT1A receptor full agonist, significantly decreased the extracellular levels of 5-HT in the raphe and prefrontal cortex. The 5-HT and other 5-HT1A receptor agonists, flesinoxan and 8-hydroxy-2- (di-n-propylamino)tetralon (8-OH-DPAT), significantly decreased the population excitatory postsynaptic potential (EPSP) in the CA3-CA1 excitatory synapse in a dose-dependent manner. The effect of 5-HT and other 5-HT1A receptor agonists accompanied the increase in paired-pulse facilitation (ppf) induced by short-interval two stimuli and were reversed by the coadministration of the 5-HT1A receptor agonist, NAN-190, but not by (+/-)-pindolol. (+/-)-pindolol also suppressed the EPSP, but this effect was not reversed by NAN-190. These results suggest that (+/-)-pindolol acts as a partial agonist to the somatodendritic 5-HT1A receptor in the raphe, whereas it may have no action on the postsynaptic 5-HT1A receptor in the hippocampus.

Allosteric nature of P2X receptor activation probed by photoaffinity labelling

PubMed Central

Bhargava, Y; Rettinger, J; Mourot, A

2012-01-01

BACKGROUND AND PURPOSE In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. EXPERIMENTAL APPROACH We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. KEY RESULTS Irradiation of the P2X2/1 receptor chimera – BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. CONCLUSIONS AND IMPLICATIONS Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding. PMID:22725669

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Differential agonist and inverse agonist profile of antipsychotics at D2L receptors coupled to GIRK potassium channels.

PubMed

Heusler, Peter; Newman-Tancredi, Adrian; Castro-Fernandez, Annabelle; Cussac, Didier

2007-03-01

The D(2) dopaminergic receptor represents a major target of antipsychotic drugs. Using the coupling of the human D(2long) (hD(2L)) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus laevis oocytes, we examined the activity of antipsychotic agents of different classes - typical, atypical, and a "new generation" of compounds, exhibiting a preferential D(2) and 5-HT(1A) receptor profile. When the hD(2L) receptor was coexpressed with GIRK channels, a series of reference compounds exhibited full agonist (dopamine, and quinpirole), partial agonist (apomorphine, (-)3-PPP, and (+)-UH232) or inverse agonist (raclopride, and L741626) properties. Sarizotan exhibited only very weak partial agonist action. At higher levels of receptor cRNA injected per oocyte, both partial agonist activity and inverse agonist properties were generally more pronounced. The inverse agonist action of L741626 was reversed by interaction with sarizotan, thus confirming the constitutive activity of wild-type hD(2L) receptors in the oocyte expression system. When antipsychotic agents were tested for their actions at the hD(2L) receptor, typical (haloperidol) as well as atypical (nemonapride, ziprasidone, and clozapine) compounds acted as inverse agonists. In contrast, among D(2)/5-HT(1A) antipsychotics, only SLV313 and F15063 behaved as inverse agonists, whilst the other members of this group (bifeprunox, SSR181507 and the recently marketed antipsychotic, aripiprazole) exhibited partial agonist properties. Thus, the X. laevis oocyte expression system highlights markedly different activity of antipsychotics at the hD(2L) receptor. These differential properties may translate to distinct therapeutic potential of these compounds.

Characterization of the discriminative stimulus effects of lorcaserin in rats.

PubMed

Serafine, Katherine M; Rice, Kenner C; France, Charles P

2016-09-01

Lorcaserin is approved by the Food and Drug Administration for treating obesity and is under consideration for treating substance use disorders; it has agonist properties at serotonin (5-HT)2C receptors and might also have agonist properties at other 5-HT receptor subtypes. This study used drug discrimination to investigate the mechanism(s) of action of lorcaserin. Male Sprague-Dawley rats discriminated 0.56 mg/kg i.p. lorcaserin from saline while responding under a fixed-ratio 5 schedule for food. Lorcaserin (0.178-1.0 mg/kg) dose-dependently increased lorcaserin-lever responding. The 5-HT2C receptor agonist mCPP and the 5-HT2A receptor agonist DOM each occasioned greater than 90% lorcaserin-lever responding in seven of eight rats. The 5-HT1A receptor agonist 8-OH-DPAT occasioned greater than 90% lorcaserin-lever responding in four of seven rats. The 5-HT2C receptor selective antagonist SB 242084 attenuated lorcaserin-lever responding in all eight rats and the 5-HT2A receptor selective antagonist MDL 100907 attenuated lorcaserin-lever responding in six of seven rats. These results suggest that, in addition to agonist properties at 5-HT2C receptors, lorcaserin also has agonist properties at 5-HT2A and 5-HT1A receptors. Because some drugs with 5-HT2A receptor agonist properties are abused, it is important to fully characterize the behavioral effects of lorcaserin while considering its potential for treating substance use disorders. © 2016 Society for the Experimental Analysis of Behavior.

Neurokinin receptor modulation of respiratory activity in the rabbit.

PubMed

Bongianni, Fulvia; Mutolo, Donatella; Cinelli, Elenia; Pantaleo, Tito

2008-06-01

The respiratory role of neurokinin (NK) receptors was investigated in alpha-chloralose-urethane-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nL) of NK receptor agonists and antagonists. Microinjections were performed in a region located just caudal to the rostral expiratory neurons. This region displayed features similar to those of the pre-Bötzinger complex (pre-BötC) of adult cats and rats, and proved to produce excitatory respiratory effects in response to microinjections of D,L-homocysteic acid. We used as agonists (0.1, 0.5 and 5 mM) substance P (SP), the NK1 receptor agonists [Sar(9), Met(O2)(11)]-SP and GR 73632, the NK2 receptor agonist NKA, the NK3 receptor agonist senktide, and as antagonists (5 mM) the NK1 receptor antagonist CP-99,994 and the NK2 receptor antagonist MEN 10376. SP always increased respiratory frequency, but NK1 receptor agonists did not change respiratory variables. NKA and senktide at 5 mm increased respiratory frequency. CP-99,994 caused increases in respiratory frequency and did not antagonize the effects of SP. MEN 10376 prevented the respiratory responses induced by NKA and reduced those provoked by SP. SP or the NK1 receptor agonists (5 mM) injected (1 microL) into the IV ventricle caused marked excitatory effects on respiration. The results suggest that NK2 and NK3, but not NK1, receptors are involved in the excitatory modulation of inspiratory activity within the investigated region and are consistent with the notion that the pre-BötC neurons are important components of the inspiratory rhythm-generating mechanisms.

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

PubMed

Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

2015-07-01

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Discrimination between olfactory receptor agonists and non-agonists.

PubMed

Topin, Jérémie; de March, Claire A; Charlier, Landry; Ronin, Catherine; Antonczak, Serge; Golebiowski, Jérôme

2014-08-11

A joint approach combining free-energy calculations and calcium-imaging assays on the broadly tuned human 1G1 olfactory receptor is reported. The free energy of binding of ten odorants was computed by means of molecular-dynamics simulations. This state function allows separating the experimentally determined eight agonists from the two non-agonists. This study constitutes a proof-of-principle for the computational deorphanization of olfactory receptors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of 3H-noradrenaline release by presynaptic opioid, cannabinoid and bradykinin receptors and β-adrenoceptors in mouse tissues

PubMed Central

Trendelenburg, A U; Cox, S L; Schelb, V; Klebroff, W; Khairallah, L; Starke, K

2000-01-01

Release-modulating opioid and cannabinoid (CB) receptors, β-adrenoceptors and bradykinin receptors at noradrenergic axons were studied in mouse tissues (occipito-parietal cortex, heart atria, vas deferens and spleen) preincubated with 3H-noradrenaline. Experiments using the OP1 receptor-selective agonists DPDPE and DSLET, the OP2-selective agonists U50488H and U69593, the OP3-selective agonist DAMGO, the ORL1 receptor-selective agonist nociceptin, and a number of selective antagonists showed that the noradrenergic axons innervating the occipito-parietal cortex possess release-inhibiting OP3 and ORL1 receptors, those innervating atria OP1, ORL1 and possibly OP3 receptors, and those innervating the vas deferens all four opioid receptor types. Experiments using the non-selective CB agonists WIN 55,212-2 and CP 55,940 and the CB1-selective antagonist SR 141716A indicated that the noradrenergic axons of the vas deferens possess release-inhibiting CB1 receptors. Presynaptic CB receptors were not found in the occipito-parietal cortex, in atria or in the spleen. Experiments using the non-selective β-adrenoceptor agonist isoprenaline and the β2-selective agonist salbutamol, as well as subtype-selective antagonists, demonstrated the occurrence of release-enhancing β2-adrenoceptors at the sympathetic axons of atria and the spleen, but demonstrated their absence in the occipito-parietal cortex and the vas deferens. Experiments with bradykinin and the B2-selective antagonist Hoe 140 showed the operation of release-enhancing B2 receptors at the sympathetic axons of atria, the vas deferens and the spleen, but showed their absence in the occipito-parietal cortex. The experiments document a number of new presynaptic receptor locations. They confirm and extend the existence of marked tissue and species differences in presynaptic receptors at noradrenergic neurons. PMID:10807669

Pharmacological characterization of the new histamine H4 receptor agonist VUF 8430

PubMed Central

Lim, Herman D; Adami, Maristella; Guaita, Elena; Werfel, Thomas; Smits, Rogier A; de Esch, Iwan JP; Bakker, Remko A; Gutzmer, Ralf; Coruzzi, Gabriella; Leurs, Rob

2009-01-01

Background and purpose: We compare the pharmacological profiles of a new histamine H4 receptor agonist 2-(2-guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H4 receptor agonist, 4-methylhistamine. Experimental approach: Radioligand binding and functional assays were performed using histamine H4 receptors expressed in mammalian cell lines. Compounds were also evaluated ex vivo in monocyte-derived dendritic cells endogenously expressing H4 receptors and in vivo in anaesthetized rats for gastric acid secretion activity. Key results: Both VUF 8430 and 4-methylhistamine were full agonists at human H4 receptors with lower affinity at rat and mouse H4 receptors. Both compounds induced chemotaxis of monocyte-derived dendritic cells. VUF 8430 also showed reasonable affinity and was a full agonist at the H3 receptor. Agmatine is a metabolite of arginine, structurally related to VUF 8430, and was a H4 receptor agonist with micromolar affinity. At histamine H3 receptors, agmatine was a full agonist, whereas 4-methylhistamine was an agonist only at high concentrations. Both VUF 8430 and agmatine were inactive at H1 and H2 receptors, whereas 4-methylhistamine is as active as histamine at H2 receptors. In vivo, VUF 8430 only caused a weak secretion of gastric acid mediated by H2 receptors, whereas 4-methylhistamine, dimaprit, histamine and amthamine, at equimolar doses, induced 2.5- to 6-fold higher output than VUF 8430. Conclusions and implications: Our results suggest complementary use of 4-methylhistamine and VUF 8430 as H4 receptor agonists. Along with H4 receptor antagonists, both agonists can serve as useful pharmacological tools in studies of histamine H4 receptors. PMID:19413569

The pharmacology of Ro 64-6198, a systemically active, nonpeptide NOP receptor (opiate receptor-like 1, ORL-1) agonist with diverse preclinical therapeutic activity.

PubMed

Shoblock, James R

2007-01-01

The NOP receptor (formerly referred to as opiate receptor-like 1, ORL-1, LC132, OP(4), or NOP(1)) is a G protein-coupled receptor that shares high homology to the classic opioid MOP, DOP, and KOP (mu, delta, and kappa, respectively) receptors and was first cloned in 1994 by several groups. The NOP receptor remained an orphan receptor until 1995, when the endogenous neuropeptide agonist, known as nociceptin or orphanin FQ (N/OFQ) was isolated. Five years later, a group at Hoffmann-La Roche reported on the selective, nonpeptide NOP agonist Ro 64-6198, which became the most extensively published nonpeptide NOP agonist and a valuable pharmacological tool in determining the potential of the NOP receptor as a therapeutic target. Ro 64-6198 is systemically active and achieves high brain penetration. It has subnanomolar affinity for the NOP receptor and is at least 100 times more selective for the NOP receptor over the classic opioid receptors. Ro 64-6198 ranges from partial to full agonist, depending on the assay. Preclinical data indicate that Ro 64-6198 may have broad clinical uses, such as in treating stress and anxiety, addiction, neuropathic pain, cough, and anorexia. This review summarizes the pharmacology and preclinical data of Ro 64-6198.

Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

PubMed

Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

2015-12-01

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice

PubMed Central

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2014-01-01

Somatostatin interacts with 5 G-protein-coupled receptor (sst1–5). Octreotide, a stable sst2≫3≥5 agonist, octreotide, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1–5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10 μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (4 sets of 3 CRD, each at 55 mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between 2 sets of graded CRD (15, 30, 45, and 60 mmHg, 3 times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60 mmHg CRD, respectively. ODT8-SST (10 μg) and the sst2 agonist, S-346-011 (3 and 10 μg) prevented mechanically-induced visceral hypersensitivity in the 3 sets of CRD, the sst1 agonist (10 μg) blocked only the 2nd set and showed a trend at 3 μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10 μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10 μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. PMID:25451334

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice.

PubMed

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2015-01-01

Somatostatin interacts with five G-protein-coupled receptor (sst1-5). Octreotide, a stable sst2≫3≥5 agonist, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1-5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (four sets of three CRD, each at 55mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between two sets of graded CRD (15, 30, 45, and 60mmHg, three times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60mmHg CRD, respectively. ODT8-SST (10μg) and the sst2 agonist, S-346-011 (3 and 10μg) prevented mechanically induced visceral hypersensitivity in the three sets of CRD, the sst1 agonist (10μg) blocked only the 2nd set and showed a trend at 3μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. Copyright © 2014 Elsevier Inc. All rights reserved.

Directly Observable Behavioral Effects of Lorcaserin in Rats.

PubMed

Serafine, Katherine M; Rice, Kenner C; France, Charles P

2015-12-01

(1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (lorcaserin) is approved by the United States Food and Drug Administration for treating obesity, and its therapeutic effects are thought to result from agonist activity at serotonin (5-HT)2C receptors. Lorcaserin has affinity for other 5-HT receptor subtypes, although its activity at those subtypes is not fully described. The current study compared the behavioral effects of lorcaserin (0.0032-32.0 mg/kg) to the effects of other 5-HT receptor selective agonists in rats (n = 8). The 5-HT2C receptor selective agonist 1-(3-chlorophenyl)piperazine (mCPP, 0.032-1.0 mg/kg) and lorcaserin induced yawning which was attenuated by the 5-HT2C receptor selective antagonist 6-chloro-5-methyl-N-(6-[(2-methylpyridin-3-yl)oxy]pydidin-3-yl)indoline-1-carboxamide (1.0 mg/kg). The 5-HT2A receptor selective agonist 2,5-dimethoxy-4-methylamphetamine (0.1-3.2 mg/kg) induced head twitching, which was attenuated by the 5-HT2A receptor selective antagonist R-(+)-2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL 100907, 0.01 mg/kg), lorcaserin (3.2 mg/kg), and mCPP (3.2 mg/kg). In rats pretreated with MDL 100907 (1.0 mg/kg), lorcaserin also induced head twitching. At larger doses, lorcaserin produced forepaw treading, which was attenuated by the 5-HT1A receptor selective antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (0.178 mg/kg). While the behavioral effects of lorcaserin in rats are consistent with it having agonist activity at 5-HT2C receptors, these data suggest that at larger doses it also has agonist activity at 5-HT2A and possibly 5-HT1A receptors. Mounting evidence suggests that 5-HT2C receptor agonists might be effective for treating drug abuse. A more complete description of the activity of lorcaserin at 5-HT receptor subtypes will facilitate a better understanding of the mechanisms that mediate its therapeutic effects. U.S. Government work not protected by U.S. copyright.

Directly Observable Behavioral Effects of Lorcaserin in Rats

PubMed Central

Serafine, Katherine M.; Rice, Kenner C.

2015-01-01

(1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (lorcaserin) is approved by the United States Food and Drug Administration for treating obesity, and its therapeutic effects are thought to result from agonist activity at serotonin (5-HT)2C receptors. Lorcaserin has affinity for other 5-HT receptor subtypes, although its activity at those subtypes is not fully described. The current study compared the behavioral effects of lorcaserin (0.0032–32.0 mg/kg) to the effects of other 5-HT receptor selective agonists in rats (n = 8). The 5-HT2C receptor selective agonist 1-(3-chlorophenyl)piperazine (mCPP, 0.032–1.0 mg/kg) and lorcaserin induced yawning which was attenuated by the 5-HT2C receptor selective antagonist 6-chloro-5-methyl-N-(6-[(2-methylpyridin-3-yl)oxy]pydidin-3-yl)indoline-1-carboxamide (1.0 mg/kg). The 5-HT2A receptor selective agonist 2,5-dimethoxy-4-methylamphetamine (0.1–3.2 mg/kg) induced head twitching, which was attenuated by the 5-HT2A receptor selective antagonist R-(+)-2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL 100907, 0.01 mg/kg), lorcaserin (3.2 mg/kg), and mCPP (3.2 mg/kg). In rats pretreated with MDL 100907 (1.0 mg/kg), lorcaserin also induced head twitching. At larger doses, lorcaserin produced forepaw treading, which was attenuated by the 5-HT1A receptor selective antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (0.178 mg/kg). While the behavioral effects of lorcaserin in rats are consistent with it having agonist activity at 5-HT2C receptors, these data suggest that at larger doses it also has agonist activity at 5-HT2A and possibly 5-HT1A receptors. Mounting evidence suggests that 5-HT2C receptor agonists might be effective for treating drug abuse. A more complete description of the activity of lorcaserin at 5-HT receptor subtypes will facilitate a better understanding of the mechanisms that mediate its therapeutic effects. PMID:26384326

D1 receptor agonist improves sleep-wake parameters in experimental parkinsonism.

PubMed

Hyacinthe, Carole; Barraud, Quentin; Tison, François; Bezard, Erwan; Ghorayeb, Imad

2014-03-01

Both excessive daytime sleepiness (EDS) and rapid eye movement (REM) sleep deregulation are part of Parkinson's disease (PD) non-motor symptoms and may complicate dopamine replacement therapy. We report here that dopamine agonists act differentially on sleep architecture in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine macaque monkey. Continuous sleep and wake electroencephalographic monitoring revealed no effect of the selective dopamine D2 receptor agonist quinpirole on EDS, whereas the selective dopamine D1 receptor agonist SKF38393 efficiently alleviated EDS and restored REM sleep to baseline values. The present results question the relevance of abandoning D1 receptor agonist treatment in PD as it might actually improve sleep-related disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

Tachykinin-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK2 receptors

PubMed Central

Kerr, Karen P; Thai, Binh; Coupar, Ian M

2000-01-01

The tachykinin receptor present in the guinea-pig oesophageal mucosa that mediates contractile responses of the muscularis mucosae has been characterized, using functional in vitro experiments. The NK1 receptor-selective agonist, [Sar9(O2)Met11]SP and the NK3 receptor-selective agonists, [MePhe7]-NKB and senktide, produced no response at submicromolar concentrations. The NK2 receptor-selective agonists, [Nle10]-NKA(4–10), and GR 64,349 produced concentration-dependent contractile effects with pD2 values of 8.20±0.16 and 8.30±0.15, respectively. The concentration-response curve to the non-selective agonist, NKA (pD2=8.13±0.04) was shifted significantly rightwards only by the NK2 receptor-selective antagonist, GR 159,897 and was unaffected by the NK1 receptor-selective antagonist, SR 140,333 and the NK3 receptor-selective antagonist, SB 222,200. The NK2 receptor-selective antagonist, GR 159,897, exhibited an apparent competitive antagonism against the NK2 receptor-selective agonist, GR 64,349 (apparent pKB value=9.29±0.16) and against the non-selective agonist, NKA (apparent pKB value=8.71±0.19). The NK2 receptor-selective antagonist, SR 48,968 exhibited a non-competitive antagonism against the NK2 receptor-selective agonist, [Nle10]-NKA(4–10). The pKB value was 10.84±0.19. It is concluded that the guinea-pig isolated oesophageal mucosa is a useful preparation for studying the effects of NK2 receptor-selective agonists and antagonists as the contractile responses to various tachykinins are mediated solely by NK2 receptors. PMID:11090121

Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor.

PubMed

Callaghan, Brid; Kosari, Samin; Pustovit, Ruslan V; Sartor, Daniela M; Ferens, Dorota; Ban, Kung; Baell, Jonathan; Nguyen, Trung V; Rivera, Leni R; Brock, James A; Furness, John B

2014-03-01

Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. © 2013 The British Pharmacological Society.

PF-06827443 Displays Robust Allosteric Agonist and Positive Allosteric Modulator Activity in High Receptor Reserve and Native Systems.

PubMed

Moran, Sean P; Cho, Hyekyung P; Maksymetz, James; Remke, Daniel H; Hanson, Ryan M; Niswender, Colleen M; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

2018-04-25

Positive allosteric modulators (PAMs) of the M 1 subtype of muscarinic acetylcholine receptor have attracted intense interest as an exciting new approach for improving the cognitive deficits in schizophrenia and Alzheimer's disease. Recent evidence suggests that the presence of intrinsic agonist activity of some M 1 PAMs may reduce efficacy and contribute to adverse effect liability. However, the M 1 PAM PF-06827443 was reported to have only weak agonist activity at human M 1 receptors but produced M 1 -dependent adverse effects. We now report that PF-06827443 is an allosteric agonist in cell lines expressing rat, dog, and human M 1 and use of inducible cell lines shows that agonist activity of PF-06827443 is dependent on receptor reserve. Furthermore, PF-06827443 is an agonist in native tissue preparations and induces behavioral convulsions in mice similar to other ago-PAMs. These findings suggest that PF-06827443 is a robust ago-PAM, independent of species, in cell lines and native systems.

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

PubMed Central

Alder, J Tracy; Hacksell, Uli; Strange, Philip G

2003-01-01

Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

Receptor-selective, peptidase-resistant agonists at neurokinin NK-1 and NK-2 receptors: new tools for investigating neurokinin function.

PubMed

Hagan, R M; Ireland, S J; Jordan, C C; Beresford, I J; Deal, M J; Ward, P

1991-06-01

The pharmacological profiles of two novel neurokinin agonists have been investigated. delta Ava[L-Pro9,N-MeLeu10]SP(7-11) (GR73632) and [Lys3,Gly8-R-gamma-lactam-Leu9] NKA(3-10) (GR64349) are potent and selective agonists at NK-1 and NK-2 receptors respectively. In the guinea-pig isolated trachea preparation, contractions induced by these agonists were largely unaffected by inclusion of peptidase inhibitors in the bathing medium, indicating that these agonists are resistant to metabolism by peptidases. In the anaesthetised guinea-pig, both agonists were more potent bronchoconstrictor agents than either NKA or the SP analogue, SP methylester. In the anaesthetised rat, the NK-1 agonist, GR73632 was more potent than SP, NKA or NKB at causing the histamine-independent extravasation of plasma proteins into the skin after intradermal administration. The NK-2 agonist, GR64349 and the NK-3 agonist, senktide were without significant effect in this model. These agonists are useful tools for characterizing neurokinin receptor-mediated actions both in vitro and in vivo.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

PubMed

Budd, D C; McDonald, J E; Tobin, A B

2000-06-30

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2

PubMed Central

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V1) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys8]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg8]-vasopressin (AVP) at V1 and vasopressin-2 (V2) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V1 and V2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [3H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V1) and cyclic adenosine monophosphate (V2). Binding potency at V1 and V2 was AVP>LVP>>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V1 than for V2. Cellular activity potency was also AVP>LVP>>terlipressin. Terlipressin was a partial agonist at V1 and a full agonist at V2; LVP was a full agonist at both V1 and V2. The in vivo response to terlipressin is likely due to the partial V1 agonist activity of terlipressin and full V1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors. PMID:29302194

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

PubMed

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

[Effects of GLP-1 receptor agonists on carbohydrate metabolism control].

PubMed

Fernández-García, José Carlos; Colomo, Natalia; Tinahones, Francisco José

2014-01-01

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptor agonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs.

Activation of neurotensin receptors and purinoceptors in human colonic adenocarcinoma cells detected with the microphysiometer.

PubMed

Richards, M; van Giersbergen, P; Zimmermann, A; Lesur, B; Hoflack, J

1997-10-01

Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may modulate receptor sequestration, whereas activation of the low agonist affinity state may be linked to cellular metabolism. Comparison of our results with published data found differences as well as similarities of NT responses among three lines of HT-29 cells.

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

PubMed Central

2012-01-01

Background Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3. Results Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect. Conclusion Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology. PMID:22651821

Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors.

PubMed

Sánchez-Más, Jesús; Guillo, Lidia A; Zanna, Paola; Jiménez-Cervantes, Celia; García-Borrón, José C

2005-04-01

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

PubMed

Ziegler, Nicole; Bätz, Julia; Zabel, Ulrike; Lohse, Martin J; Hoffmann, Carsten

2011-02-01

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Structure-activity relationship of 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole analogues as 5-HT(6) receptor agonists.

PubMed

Mattsson, Cecilia; Svensson, Peder; Boettcher, Henning; Sonesson, Clas

2013-05-01

To further investigate the structure-activity relationship (SAR) of the 5-hydroxytryptamine type 6 (5-HT6) receptor agonist 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (EMD386088, 6), a series of 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were synthesized, and in vitro affinity to, and functional activity at 5-HT6 receptors was tested. We focused on substituents made at the indole N(1)-, 2- and 5-positions and these were found to not only influence the affinity at 5-HT6 receptors but also the intrinsic activity leading to antagonists, partial agonists and full agonists. In order for a compound to demonstrate potent 5-HT6 receptor agonist properties, the indole N(1) should be unsubstituted, an alkyl group such as 2-methyl is needed and finally halogen substituents in the indole 5-position (fluoro, chloro or, bromo) were essential requirements. However, the introduction of a benzenesulfonyl group at N(1)-position switched the full agonist 6 to be a 5-HT6 receptor antagonist (30). A few compounds within the 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were also screened for off-targets and generally they displayed low affinity for other 5-HT subtypes and serotonin transporter protein (SERT). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor

PubMed Central

Callaghan, Brid; Kosari, Samin; Pustovit, Ruslan V; Sartor, Daniela M; Ferens, Dorota; Ban, Kung; Baell, Jonathan; Nguyen, Trung V; Rivera, Leni R; Brock, James A; Furness, John B

2014-01-01

BACKGROUND AND PURPOSE Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. PMID:24670149

Opioid-receptor subtype agonist-induced enhancements of sucrose intake are dependent upon sucrose concentration.

PubMed

Ruegg, H; Yu, W Z; Bodnar, R J

1997-07-01

Selective mu ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO)), delta1 ([D-Pen2, D-Pen5]-enkephalin (DPDPE)), delta2 ([D-Ala2, Glu4]-Deltorphin (Delt II)), kappa1 (U50488H) and kappa3 (naloxone benzoylhydrazone (NalBzOH)) opioid agonists each stimulate food intake in rats. Whereas studies with selective opioid antagonists implicate mu and kappa1 receptors in the mediation of sucrose intake, studies with selective opioid agonists implicate mu and delta receptors in the mediation of saccharin intake. The present study determined if specific delta1, delta2, kappa1, kappa3 and mu opioid-receptor subtype agonists produced similar alterations in sucrose intake as a function of sucrose concentration (0.5%, 2.5%, 10%) across a 1-h time-course. Each of these agonists significantly increased sucrose intake with variations in pattern, magnitude, and consistency as a function of sucrose concentration. Whereas the mu opioid agonist, DAMGO, and the delta1 opioid agonist, DPDPE, each enhanced sucrose intake at higher (2.5%, 10%), but not lower (0.5%), concentrations, the delta2 opioid agonist, Delt II, increased sucrose intake at lower (0.5%, 2.5%), but not higher (10%), concentrations. Kappa opioid agonists produced less consistent effects. The kappa1 opioid agonist, U50488H, increased sucrose intake at high (10%) concentrations and decreased sucrose intake at low (0.5%) concentrations, and the kappa3 opioid agonist, NalBzOH, inconsistently increased sucrose intake at the 0.5% (20 microg) and 10% (1 microg) concentrations. Thus, these data further implicate mu, delta1, and delta2 opioid mediation of palatable intake, particularly of its orosensory characteristics.

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes.

PubMed

Newman-Tancredi, Adrian; Cussac, Didier; Quentric, Yann; Touzard, Manuelle; Verrièle, Laurence; Carpentier, Nathalie; Millan, Mark J

2002-11-01

Although certain antiparkinson agents interact with serotonin (5-HT) receptors, little information is available concerning functional actions. Herein, we characterized efficacies of apomorphine, bromocriptine, cabergoline, lisuride, piribedil, pergolide, roxindole, and terguride at human (h)5-HT(1A), h5-HT(1B), and h5-HT(1D) receptors [guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding], and at h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors (depletion of membrane-bound [(3)H]phosphatydilinositol). All drugs stimulated h5-HT(1A) receptors with efficacies (compared with 5-HT, 100%) ranging from modest (apomorphine, 35%) to high (cabergoline, 93%). At h5-HT(1B) receptors, efficacies varied from mild (terguride, 37%) to marked (cabergoline, 102%) and potencies were modest (pEC(50) values of 5.8-7.6): h5-HT(1D) sites were activated with a similar range of efficacies and greater potency (7.1-8.5). Piribedil and apomorphine were inactive at h5-HT(1B) and h5-HT(1D) receptors. At h5-HT(2A) receptors, terguride, lisuride, bromocriptine, cabergoline, and pergolide displayed potent (7.6-8.8) agonist properties (49-103%), whereas apomorphine and roxindole were antagonists and piribedil was inactive. Only pergolide (113%/8.2) and cabergoline (123%/8.6) displayed pronounced agonist properties at h5-HT(2B) receptors. At 5-HT(2C) receptors, lisuride, bromocriptine, pergolide, and cabergoline were efficacious (75-96%) agonists, apomorphine and terguride were antagonists, and piribedil was inactive. MDL100,907 and SB242,084, selective antagonists at 5-HT(2A) and 5-HT(2C) receptors, respectively, abolished these actions of pergolide, cabergoline, and bromocriptine. In conclusion, antiparkinson agents display markedly different patterns of agonist and antagonist properties at multiple 5-HT receptor subtypes. Although all show modest (agonist) activity at 5-HT(1A) sites, their contrasting actions at 5-HT(2A) and 5-HT(2C) sites may be of particular significance to their functional profiles in vivo.

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

PubMed Central

Tulapurkar, M E; Laubinger, W; Nahum, V; Fischer, B; Reiser, G

2004-01-01

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-α-B) were synthesized by substitution of a nonbridging O at Pα with a BH3 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 P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1-GFP, rP2Y2-GFP). We investigated agonist-induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y1-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-α-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 Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP-α-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 P2Y1 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. PMID:15197109

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

PubMed

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

2011-04-14

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Effects of Trace Amine-associated Receptor 1 Agonists on the Expression, Reconsolidation, and Extinction of Cocaine Reward Memory.

PubMed

Liu, Jian-Feng; Thorn, David A; Zhang, Yanan; Li, Jun-Xu

2016-07-01

As a modulator of dopaminergic system, trace amine-associated receptor 1 has been shown to play a critical role in regulating the rewarding properties of additive drugs. It has been demonstrated that activation of trace amine-associated receptor 1 decreased the abuse-related behaviors of cocaine in rats. However, the role of trace amine-associated receptor 1 in specific stages of cocaine reward memory is still unclear. Here, using a cocaine-induced conditioned place preference model, we tested the effects of a selective trace amine-associated receptor 1 agonist RO5166017 on the expression, reconsolidation, and extinction of cocaine reward memory. We found that RO5166017 inhibited the expression but not retention of cocaine-induced conditioned place preference. RO5166017 had no effect on the reconsolidation of cocaine reward memory. Pretreatment with RO5166017 before extinction hindered the formation of extinction long-term memory. RO5166017 did not affect the movement during the conditioned place preference test, indicating the inhibitory effect of RO5166017 on the expression of cocaine-induced conditioned place preference was not caused by locomotion inhibition. Using a cocaine i.v. self-administration model, we found that the combined trace amine-associated receptor 1 partial agonist RO5263397 with extinction had no effect on the following cue- and drug-induced reinstatement of cocaine-seeking behavior. Repeated administration of the trace amine-associated receptor 1 agonist during extinction showed a continually inhibitory effect on the expression of cocaine reward memory both in cocaine-induced conditioned place preference and cocaine self-administration models. Taken together, these results indicate that activation of trace amine-associated receptor 1 specifically inhibited the expression of cocaine reward memory. The inhibitory effect of trace amine-associated receptor 1 agonists on cocaine reward memory suggests that trace amine-associated receptor 1 agonists could be a promising agent to prevent cocaine relapse. © The Author 2016. Published by Oxford University Press on behalf of CINP.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

PubMed

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Synthesis and pharmacological characterization of beta2-adrenergic agonist enantiomers: zilpaterol.

PubMed

Kern, Christopher; Meyer, Thorsten; Droux, Serge; Schollmeyer, Dieter; Miculka, Christian

2009-03-26

The beta-adrenergic agonist 1 (zilpaterol) is used as production enhancer in cattle. Binding experiments of separated enantiomers on recombinant human beta(2)-adrenergic and mu-opioid receptors and functional studies showed that the (-)-1 enantiomer accounts for essentially all the beta(2)-adrenergic agonist activity and that it exhibits less affinity toward the mu-opioid receptor than (+)-1, which is a mu-opioid receptor antagonist. X-ray crystallography revealed the absolute configuration of (-)-1 to be 6R,7R.

A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

PubMed

Fay, Jonathan F; Farrens, David L

2012-09-28

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.

D-1 and D-2 receptor blockade have additive cataleptic effects in mice, but receptor effects may interact in opposite ways.

PubMed

Klemm, W R; Block, H

1988-02-01

The dopaminergic role of D-1 and D-2 receptors in catalepsy was evaluated using drugs with preferential receptor affinities. The D-1 antagonist, SCH 23390, caused distinct catalepsy in mice at 1, 2, and 10 mg/kg, IP, but not at two lower doses. The selective D-1 blocker, molindone, also caused catalepsy at 5 and 10 mg/kg; and blockade of both receptor types produced additive cataleptogenic effects. Apomorphine (4 mg/kg), which is an agonist for both receptors, potentiated SCH 23390-induced catalepsy much more than it did the catalepsy induced by molindone; the potentiation was produced by higher, not lower, doses of apomorphine. To determine if the apomorphine potentiation was mediated by D-1 or D-2 receptors, we tested selective agonists in mice that were concurrently injected with selective blockers. SCH 23390-induced catalepsy was potentiated by a large dose of the D-2 agonist, bromocriptine. The catalepsy of D-2 blockade with molindone was not potentiated by the D-1 agonist, SKF 38393, which slightly disrupted the catalepsy of D-2 blockade. We conclude that catalepsy is not a simple D-2 blockade phenomenon and that preferential antagonism of either receptor type can cause catalepsy. Catalepsy is most profound when both receptor types are blocked. Dopamine agonists, in large concentrations, are known to promote movements, and thus it is not surprising that they tend to disrupt catalepsy.(ABSTRACT TRUNCATED AT 250 WORDS)

An ionotropic but not a metabotropic glutamate agonist potentiates the pharmacological effects of olanzapine in the rat.

PubMed

Dall'Olio, Rossella; Rimondini, Roberto; Locchi, Federica; Voltattorni, Manuela; Gandolfi, Ottavio

2005-12-01

This study aimed to evaluate the possible potentiating action of ionotropic or metabotropic (metabotropic glutamate receptor type 5) glutamate agonists on pharmacological effects induced in rats by the atypical antipsychotic olanzapine. The administration of doses of olanzapine, which did not affect spontaneous motility, inhibited behaviors induced by the selective stimulation of 5HT(2A) and D(2) receptors. In particular, 0.03 or 0.06 mg/kg of olanzapine was sufficient to reduce, respectively, head shakes induced by the 5HT(2A) agonist 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (1 mg/kg) or hypermotility elicited by the D(2) stimulant quinpirole (0.15 mg/kg). Behavioral responses to a D(1)/D(2) agonist (apomorphine-induced stereotypies) were inhibited by doses of olanzapine that also influenced spontaneous behavior. The concomitant administration of D-cycloserine, an agonist at the glycine site on the N-methyl-D-aspartate receptor complex, given at a dose (3 mg/kg) that did not affect behavior, increased the inhibitory effect of olanzapine on the responses produced by 5HT2A, D(2) and D(1)/D(2) receptor stimulation. The concomitant administration of 2-chloro-5-hydroxyphenylglycine, an agonist of metabotropic glutamate receptor type 5, increased the inhibitory effect of olanzapine on the behaviors induced by the stimulation of D(2), but not 5HT2A or D(1)/D(2) receptors. As the effect on the serotonergic system seems important for the unusual pharmacological profile of atypical antipsychotics, the present results suggest that N-methyl-D-aspartate, but not metabotropic glutamate receptor type 5 agonists could be seen as promising therapeutic agents for increasing the pharmacological effects of olanzapine.

The effect of various opiate receptor agonists on the seizure threshold in the rat. Is dynorphin an endogenous anticonvulsant?

PubMed

Przewłocka, B; Stala, L; Lasoń, W; Przewłocki, R

1983-01-01

The effects of various opiate receptor agonists on the seizure threshold after an intravenous infusion of pentylenetetrazol were investigated in rats. The mu- and epsilon-receptor agonists, morphine (20-40 micrograms) and beta-endorphin (5-10 micrograms) show proconvulsant properties towards clonic and tonic seizures. The delta-receptor agonist (D-Ala2,D-Leu5-enkephalin, DADL 5-40 micrograms) and alpha-neoendorphin (20-40 micrograms) show pro- and anticonvulsant properties towards clonic and tonic seizures, respectively. Anticonvulsant properties of DADL are possibly due to its action on the spinal cord, since after the intrathecal injection this effect is still observed. Similarities between DADL and alpha-neoendorphin suggest that they may act through the same receptor. The kappa-receptor agonist dynorphin A (5-20 micrograms) and its degradation-resistant analogue D-Arg-dynorphin1-13 (10 micrograms) show significant anticonvulsant properties. Our present results suggest that the kappa-receptor agonist dynorphin may act physiologically as an endogenous anticonvulsant, in contrast to other opioid peptides.

[Effects of GLP-1 receptor agonists on carbohydrate metabolism control].

PubMed

Fernández-García, José Carlos; Colomo, Natalia; Tinahones, Francisco José

2014-09-01

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptor agonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

5-Amino-6-chloro-N-[(1-isobutylpiperidin-4-yl)methyl]-2-methylimidazo[1,2-alpha]pyridine-8-carboxamide (CJ-033,466), a novel and selective 5-hydroxytryptamine4 receptor partial agonist: pharmacological profile in vitro and gastroprokinetic effect in conscious dogs.

PubMed

Mikami, Tadayoshi; Ochi, Yasuo; Suzuki, Keiko; Saito, Toshiyuki; Sugie, Yutaka; Sakakibara, Minoru

2008-04-01

5-Hydroxytryptamine (5-HT) receptors and dopamine(2) (D(2)) receptor modulate gastrointestinal motility. Gastroprokinetic agents that act on several 5-HT receptor subtypes and/or D(2) receptors are used clinically. Although the 5-HT(4) receptor is known to mediate the gastroprokinetic effects of these agents, the absence of highly selective 5-HT(4) receptor agonists has made it difficult to confirm the physiological consequences of selective 5-HT(4) receptor stimulation. In this study, we report the in vitro pharmacological profiles and the in vivo gastroprokinetic effects of 5-amino-6-chloro-N-[(1-isobutylpiperidin-4-yl)methyl]-2-methylimidazo[1,2-alpha]pyridine-8-carboxamide (CJ-033,466), a novel, potent, and selective 5-HT(4) partial agonist. Compared with preceding 5-HT(4) agonists such as cisapride, mosapride, and tegaserod, CJ-033,466 had a superior in vitro profile, with nanomolar agonistic activities for the 5-HT(4) receptor and 1000-fold greater selectivity for the 5-HT(4) receptor over other 5-HT and D(2) receptors. In vivo studies in conscious dogs showed that CJ-033,466 dose-dependently stimulated gastric antral motility in both the fasted and postprandial states at the same dose range and that it was 30 times more potent than cisapride. Furthermore, CJ-033,466 accelerated the gastric emptying rate in a gastroparesis dog model at the minimally effective dose established in the gastric motility study. In conclusion, CJ-033,466 is a potent and highly selective 5-HT(4) agonist that stimulates physiologically coordinated gastric motility, and it has no activity on other 5-HT receptor subtypes and D(2) receptors. Therefore, CJ-033,466 could be used to treat gastroparesis, providing better gastroprokinetics and reduced side effects mediated by the other receptors.

Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

PubMed Central

Sloop, Kyle W.; Willard, Francis S.; Brenner, Martin B.; Ficorilli, James; Valasek, Kathleen; Showalter, Aaron D.; Farb, Thomas B.; Cao, Julia X.C.; Cox, Amy L.; Michael, M. Dodson; Gutierrez Sanfeliciano, Sonia Maria; Tebbe, Mark J.; Coghlan, Michael J.

2010-01-01

OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization. PMID:20823098

Central nervous system neuropeptide Y signaling via the Y1 receptor partially dissociates feeding behavior from lipoprotein metabolism in lean rats.

PubMed

Rojas, Jennifer M; Stafford, John M; Saadat, Sanaz; Printz, Richard L; Beck-Sickinger, Annette G; Niswender, Kevin D

2012-12-15

Elevated plasma triglyceride (TG) levels contribute to an atherogenic dyslipidemia that is associated with obesity, diabetes, and metabolic syndrome. Numerous models of obesity are characterized by increased central nervous system (CNS) neuropeptide Y (NPY) tone that contributes to excess food intake and obesity. Previously, we demonstrated that intracerebroventricular (icv) administration of NPY in lean fasted rats also elevates hepatic production of very low-density lipoprotein (VLDL)-TG. Thus, we hypothesize that elevated CNS NPY action contributes to not only the pathogenesis of obesity but also dyslipidemia. Here, we sought to determine whether the effects of NPY on feeding and/or obesity are dissociable from effects on hepatic VLDL-TG secretion. Pair-fed, icv NPY-treated, chow-fed Long-Evans rats develop hypertriglyceridemia in the absence of increased food intake and body fat accumulation compared with vehicle-treated controls. We then modulated CNS NPY signaling by icv injection of selective NPY receptor agonists and found that Y1, Y2, Y4, and Y5 receptor agonists all induced hyperphagia in lean, ad libitum chow-fed Long-Evans rats, with the Y2 receptor agonist having the most pronounced effect. Next, we found that at equipotent doses for food intake NPY Y1 receptor agonist had the most robust effect on VLDL-TG secretion, a Y2 receptor agonist had a modest effect, and no effect was observed for Y4 and Y5 receptor agonists. These findings, using selective agonists, suggest the possibility that the effect of CNS NPY signaling on hepatic VLDL-TG secretion may be relatively dissociable from effects on feeding behavior via the Y1 receptor.

The effects of BMY-14802 against L-DOPA- and dopamine agonist-induced dyskinesia in the hemiparkinsonian rat

PubMed Central

Bhide, Nirmal; Lindenbach, David; Surrena, Margaret A.; Goldenberg, Adam A.; Bishop, Christopher; Berger, S. Paul; Paquette, Melanie A.

2013-01-01

Rationale L-DOPA continues to be the primary treatment for patients with Parkinson’s disease; however, the benefits of long-term treatment are often accompanied by debilitating side effects known as dyskinesias. In recent years, several 5-HT1A receptor agonists have been found to reduce dyskinesia in clinical and experimental models of PD. The purported sigma-1 antagonist, BMY-14802 has been previously demonstrated to reduce L-DOPA induced dyskinesia in a 5-HT1A receptor dependent manner. Objective In the present study, we extend these findings by examining the anti-dyskinetic potential of BMY-14802 against L-DOPA, the D1 receptor agonist SKF81297 and the D2 receptor agonist, Quinpirole, in the hemi-parkinsonian rat model. In addition, the receptor specificity of BMY-14802’s effects was evaluated using WAY-100635, a 5-HT1A receptor antagonist. Results Results confirmed the dose-dependent (20>10>5 mg/kg) anti-dyskinetic effects of BMY-14802 against L-DOPA with preservation of antiparkinsonian efficacy at 10 mg/kg. BMY-14802 at 10 and 20 mg/kg also reduced dyskinesia induced by both D1 and D2 receptor agonists. Additionally, BMY-14802’s anti-dyskinetic effects against L-DOPA, but not SKF81297 or Quinpirole, were reversed by WAY-100635 (0.5 mg/kg). Conclusion Collectively, these findings demonstrate that BMY-14802 provides anti-dyskinetic relief against L-DOPA and direct DA agonist in a preclinical model of PD, acting via multiple receptor systems and supports the utility of such compounds for the improved treatment of PD. PMID:23389756

Pain-relieving prospects for adenosine receptors and ectonucleotidases

PubMed Central

Zylka, Mark J.

2010-01-01

Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine. PMID:21236731

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery

PubMed Central

Fresco, Paula; Diniz, Carmen; Queiroz, Glória; Gonçalves, Jorge

2002-01-01

Interactions between A2A-adenosine receptors and α2-, A1- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [3H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz).Blockade of α2-autoreceptors with yohimbine (1 μM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz.The selective A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680; 1 – 100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A1-receptor agonist N6-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 μM).CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A1-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 μM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow.Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow.It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A2A-adenosine receptors is favoured by activation of release inhibitory receptors. PMID:12010771

Tachykinin receptors in the guinea-pig isolated bronchi.

PubMed

Maggi, C A; Patacchini, R; Quartara, L; Rovero, P; Santicioli, P

1991-05-17

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.

Identification of novel selective V2 receptor non-peptide agonists.

PubMed

Del Tredici, Andria L; Vanover, Kim E; Knapp, Anne E; Bertozzi, Sine M; Nash, Norman R; Burstein, Ethan S; Lameh, Jelveh; Currier, Erika A; Davis, Robert E; Brann, Mark R; Mohell, Nina; Olsson, Roger; Piu, Fabrice

2008-10-30

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

Selective and non-selective OT receptor agonists induce different locomotor behaviors in male rats via central OT receptors and peripheral V1a receptors.

PubMed

Wolfe, Monica; Wisniewska, Halina; Tariga, Hiroe; Ibanez, Gerardo; Collins, James C; Wisniewski, Kazimierz; Qi, Steve; Srinivasan, Karthik; Hargrove, Diane; Lindstrom, Beatriz Fioravanti

2018-05-21

Oxytocin (OT) continues to inspire much research due to its diverse physiological effects. While the best-understood actions of OT are uterine contraction and milk ejection, OT is also implicated in maternal and bonding behaviors, and potentially in CNS disorders such as autism, schizophrenia, and pain. The dissection of the mechanism of action of OT is complicated by the fact that this peptide activates not only its cognate receptor but also vasopressin type 1a (V1a) receptors. In this study, we evaluated OT and a selective OT receptor (OTR) agonist, FE 204409, in an automated assay that measures rat locomotor activity. The results showed: 1) Subcutaneous (sc) administration of OT decreased locomotor behavior (distance traveled, stereotypy, and rearing). This effect was reversed by a V1a receptor (V1aR) antagonist ([Pmp1,Tyr(ME)2]AVP, sc), suggesting that OT acts through peripheral V1aR to inhibit locomotor activity. 2) A selective OTR agonist (FE 204409, sc) increased stereotypy. This effect was reversed by an OTR antagonist dosed icv, suggesting a central OTR site of action. Our findings identify distinct behavioral effects for OT and the selective agonist FE 204409, adding to the growing body of evidence that the V1aR mediates many effects attributed to OT and that peptides administered systemically at supra-physiological doses may activate receptors in the brain. Our studies further emphasize the importance of utilizing selective agonists and antagonists to assess therapeutic indications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Impaired β-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor.

PubMed

Gray, David L; Allen, John A; Mente, Scot; O'Connor, Rebecca E; DeMarco, George J; Efremov, Ivan; Tierney, Patrick; Volfson, Dmitri; Davoren, Jennifer; Guilmette, Edward; Salafia, Michelle; Kozak, Rouba; Ehlers, Michael D

2018-02-14

Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of β-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of β-arrestin to D1Rs.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

PubMed

Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

2003-12-01

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Evidence for a role of 5-HT2C receptors in the motor aspects of performance, but not the efficacy of food reinforcers, in a progressive ratio schedule.

PubMed

Bezzina, G; Body, S; Cheung, T H C; Hampson, C L; Bradshaw, C M; Glennon, J C; Szabadi, E

2015-02-01

5-Hydroxytryptamine2C (5-HT2C) receptor agonists reduce the breakpoint in progressive ratio schedules of reinforcement, an effect that has been attributed to a decrease of the efficacy of positive reinforcers. However, a reduction of the breakpoint may also reflect motor impairment. Mathematical models can help to differentiate between these processes. The effects of the 5-HT2C receptor agonist Ro-600175 ((αS)-6-chloro-5-fluoro-α-methyl-1H-indole-1-ethanamine) and the non-selective 5-HT receptor agonist 1-(m-chlorophenyl)piperazine (mCPP) on rats' performance on a progressive ratio schedule maintained by food pellet reinforcers were assessed using a model derived from Killeen's Behav Brain Sci 17:105-172, 1994 general theory of schedule-controlled behaviour, 'mathematical principles of reinforcement'. Rats were trained under the progressive ratio schedule, and running and overall response rates in successive ratios were analysed using the model. The effects of the agonists on estimates of the model's parameters, and the sensitivity of these effects to selective antagonists, were examined. Ro-600175 and mCPP reduced the breakpoint. Neither agonist significantly affected a (the parameter expressing incentive value), but both agonists increased δ (the parameter expressing minimum response time). The effects of both agonists could be attenuated by the selective 5-HT2C receptor antagonist SB-242084 (6-chloro-5-methyl-N-{6-[(2-methylpyridin-3-yl)oxy]pyridin-3-yl}indoline-1-carboxamide). The effect of mCPP was not altered by isamoltane, a selective 5-HT1B receptor antagonist, or MDL-100907 ((±)2,3-dimethoxyphenyl-1-(2-(4-piperidine)methanol)), a selective 5-HT2A receptor antagonist. The results are consistent with the hypothesis that the effect of the 5-HT2C receptor agonists on progressive ratio schedule performance is mediated by an impairment of motor capacity rather than by a reduction of the incentive value of the food reinforcer.

Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

PubMed Central

Rondard, Philippe; Huang, Siluo; Monnier, Carine; Tu, Haijun; Blanchard, Bertrand; Oueslati, Nadia; Malhaire, Fanny; Li, Ying; Trinquet, Eric; Labesse, Gilles; Pin, Jean-Philippe; Liu, Jianfeng

2008-01-01

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation. PMID:18388862

Adenosine-A1 Receptor Agonist Induced Hyperalgesic Priming Type II

PubMed Central

Araldi, Dioneia; Ferrari, Luiz F.; Levine, Jon D.

2016-01-01

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) induces a model of the transition to chronic pain that we have termed Type II hyperalgesic priming. Similar to Type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, Type II hyperalgesic priming differs from Type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N6-Cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced Type II hyperalgesic priming. In this study we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced Type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the Type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor. PMID:26588695

The ventral tegmental area as a putative target for tachykinins in cardiovascular regulation

PubMed Central

Deschamps, Kathleen; Couture, Réjean

2005-01-01

Tachykinin receptor agonists and antagonists were microinjected into the ventral tegmental area (VTA) to study the relative participation of the three tachykinin receptors in cardiovascular regulation in freely behaving rat. Selective agonists (1–100 pmol) for NK1 ([Sar9, Met (O2)11]SP), NK2 ([β-Ala8]NKA (4–10)) and NK3 (senktide) receptors evoked increases in blood pressure, heart rate (HR) along with behavioural manifestations (face washing, sniffing, head scratching, rearing, wet dog shake). At 1 pmol, NK1 and NK3 agonists did not affect behaviour and blood pressure but only HR. Tachykinin agonists-induced cardiovascular responses were selectively and reversibly blocked by the prior injection of antagonists for NK1 receptors (LY 303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane), 5 nmol), NK2 receptors (SR 48968 ([(S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino-2-(3,4-dichlorophenyl)butyl]benzamide]), 250 pmol) and NK3 receptors (SB 235375 ((−)-(S)-N-(α-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide), 25 nmol). With the exception of the NK2 agonist, most behavioural effects were also blocked by antagonists. Tachykinin agonists-induced cardiovascular responses were inhibited by intravenous (i.v.) treatments with antagonists for D1 dopamine receptor (SCH23390, 0.2 mg kg−1) and β1-adrenoceptor (atenolol, 5 mg kg−1) but not for D2 dopamine receptor (raclopride, 0.16 mg kg−1). Behavioural responses were blocked by SCH23390 only. The present study provides the first pharmacological evidence that the three tachykinin receptors in the rat VTA can affect the autonomic control of blood pressure and HR by increasing midbrain dopaminergic transmission. This mechanism may be involved in the coordination of behavioural and cardiovascular responses to stress and noxious stimulation. PMID:15895109

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

PubMed Central

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N.; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W.; Broeck, Jozef Vanden; Tourwé, Dirk

2011-01-01

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity. PMID:21413804

Novel agonists for serotonin 5-HT7 receptors reverse metabotropic glutamate receptor-mediated long-term depression in the hippocampus of wild-type and Fmr1 KO mice, a model of Fragile X Syndrome

PubMed Central

Costa, Lara; Sardone, Lara M.; Lacivita, Enza; Leopoldo, Marcello; Ciranna, Lucia

2015-01-01

Serotonin 5-HT7 receptors are expressed in the hippocampus and modulate the excitability of hippocampal neurons. We have previously shown that 5-HT7 receptors modulate glutamate-mediated hippocampal synaptic transmission and long-term synaptic plasticity. In particular, we have shown that activation of 5-HT7 receptors reversed metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD) in wild-type (wt) and in Fmr1 KO mice, a mouse model of Fragile X Syndrome in which mGluR-LTD is abnormally enhanced, suggesting that 5-HT7 receptor agonists might be envisaged as a novel therapeutic strategy for Fragile X Syndrome. In this perspective, we have characterized the basic in vitro pharmacokinetic properties of novel molecules with high binding affinity and selectivity for 5-HT7 receptors and we have tested their effects on synaptic plasticity using patch clamp on acute hippocampal slices. Here we show that LP-211, a high affinity selective agonist of 5-HT7 receptors, reverses mGluR-LTD in wt and Fmr1 KO mice, correcting a synaptic malfunction in the mouse model of Fragile X Syndrome. Among novel putative agonists of 5-HT7 receptors, the compound BA-10 displayed improved affinity and selectivity for 5-HT7 receptors and improved in vitro pharmacokinetic properties with respect to LP-211. BA-10 significantly reversed mGluR-LTD in the CA3-CA1 synapse in wt and Fmr1KO mice, indicating that BA-10 behaved as a highly effective agonist of 5-HT7 receptors and reduced exaggerated mGluR-LTD in a mouse model of Fragile X Syndrome. On the other side, the compounds RA-7 and PM-20, respectively arising from in vivo metabolism of LP-211 and BA-10, had no effect on mGluR-LTD thus did not behave as agonists of 5-HT7 receptors in our conditions. The present results provide information about the structure-activity relationship of novel 5-HT7 receptor agonists and indicate that LP-211 and BA-10 might be used as novel pharmacological tools for the therapy of Fragile X Syndrome. PMID:25814945

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

PubMed

Bruins Slot, Liesbeth A; Palmier, Christiane; Tardif, Stéphanie; Cussac, Didier

2007-08-01

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

PubMed Central

Farroni, Jeffrey S; McCool, Brian A

2004-01-01

Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. PMID:15301692

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.

PubMed

Farroni, Jeffrey S; McCool, Brian A

2004-08-09

Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

Gingerols: a novel class of vanilloid receptor (VR1) agonists

PubMed Central

Dedov, Vadim N; Tran, Van H; Duke, Colin C; Connor, Mark; Christie, MacDonald J; Mandadi, Sravan; Roufogalis, Basil D

2002-01-01

Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin-activated VR1 (vanilloid) receptor. [6]-Gingerol and [8]-gingerol evoked capsaicin-like intracellular Ca2+ transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor. PMID:12411409

Identification of Buctopamine and Mebuctopamine, a β2 Receptor Agonist and Its Metabolite, in Swine Hair and Feed Additives.

PubMed

Chen, Ying-Heng; Yang, Chia-Ying; Cheng, Chih Wen; Lin, Yi-Ying; Kuo, Su Lien; Hsin, Ling-Wei

2017-05-17

4-[2-(t-Butylamino)-1-hydroxyethyl]phenol (buctopamine, 4), a new β 2 receptor agonist (β 2 -agonist), was found to be an adulterant in feed additives for swine in Taiwan, where using β 2 -agonists in food-production animals is prohibited. Buctopamine and its metabolite, 4-[2-(t-butylamino)-1-hydroxyethyl]-2-methoxyphenol (mebuctopamine, 2), were detected in swine hair specimens. Authentic compounds 2 and 4 were synthesized with 98.6% and 97.7% purity, respectively, as reference standards for analysis, and both compounds were more hydrophilic than ractopamine and clenbuterol. In a preliminary pharmacological evaluation, compounds 2 and 4 exhibited moderate human β 2 receptor binding affinity and did not show significant affinities for the human α 1 , α 2 , β 1 , and β 3 receptors. After addition of compounds 2-4 into the β 2 -agonist library, a multiresidue analysis of 26 β 2 -agonists by using triple quadrupole LC/MS/MS for routine screening conducted by regulatory authorities was established, in which the common limits of quantification for the 26 β 2 -agonists in swine feed and hair are 10 and 25 ng/g, respectively. In addition, the illegal use of buctopamine (4) has been effectively prevented. The results of this study are also useful for controlling the illegal use of new β 2 -agonists in food-production animals.

Structure and dynamics of a constitutively active neurotensin receptor

DOE PAGES

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; ...

2016-12-07

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Structure and dynamics of a constitutively active neurotensin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Structure and dynamics of a constitutively active neurotensin receptor

PubMed Central

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; Botos, Istvan; White, Courtney F.; Du, Haijuan; Vaidehi, Nagarajan; Grisshammer, Reinhard

2016-01-01

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist. PMID:27924846

75 FR 50768 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-17

.... N-Methanocarba Adenosine Derivatives and Their Dendrimer Conjugates as A 3 Receptor Agonists... receptors and agonists and antagonists of P2Y receptors, such as P2Y 1 and P2Y 14 . Dendrimer conjugates may...

An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons

PubMed Central

Hayashi, Yoshinori; Morinaga, Saori; Liu, Xia; Zhang, Jing; Wu, Zhou; Yokoyama, Takeshi; Nakanishi, Hiroshi

2016-01-01

Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca2+-activated K+ (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5′-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons. PMID:27298516

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

PubMed Central

Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

2011-01-01

BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding. PMID:20958290

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

PubMed

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Neurophysiological Analysis of Circadian Rhythm Entrainment

DTIC Science & Technology

1994-05-24

on activations in response to the ionotropic agonist NMDA (25). These results point to a previously uninvestigated role for metabotropic receptors in...are selective agonists of the metabotropic type of glutamate receptor . Selective metabotropic antagonists can block these effects but do not alter...glutamate receptors on SCN cell activity. In slice preparations, our initial findings are that the selective metabotropic agonist 1S,3R-ACPD has very potent

Role of D2 dopamine receptors of the ventral pallidum in inhibitory avoidance learning.

PubMed

Lénárd, László; Ollmann, Tamás; László, Kristóf; Kovács, Anita; Gálosi, Rita; Kállai, Veronika; Attila, Tóth; Kertes, Erika; Zagoracz, Olga; Karádi, Zoltán; Péczely, László

2017-03-15

In our present experiments, the role of D2 dopamine (DA) receptors of the ventral pallidum (VP) was investigated in one trial step-through inhibitory avoidance paradigm. Animals were shocked 3 times in the conditioning trial, with 0.5mA current for 1s. Subsequently bilateral microinjection of the D2 DA receptor agonist quinpirole was administered into the VP in three doses (0.1μg, 1.0μg or 5.0μg in 0.4μl saline). We also applied the D2 DA receptor antagonist sulpiride (0.4μg in 0.4μl saline) alone or 15min prior to the agonist treatment to elucidate whether the agonist effect was specific for the D2 DA receptors. Control animals received saline. In a supplementary experiment, it was also investigated whether application of the same conditioning method leads to the formation of short-term memory in the experimental animals. In the experiment with the D2 DA receptor agonist, only the 0.1μg quinpirole increased significantly the step-through latency during the test trials: retention was significant compared to the controls even 2 weeks after conditioning. The D2 DA receptor antagonist sulpiride pretreatment proved that the effect was due to the agonist induced activation of the D2 DA receptors of the VP. The supplementary experiment demonstrated that short-term memory is formed after conditioning in the experimental animals, supporting that the agonist enhanced memory consolidation in the first two experiments. Our results show that the activation of the D2 DA receptors in the VP facilitates memory consolidation as well as memory-retention in inhibitory avoidance paradigm. Copyright © 2017 Elsevier B.V. All rights reserved.

Elevated GnRH receptor expression plus GnRH agonist treatment inhibits the growth of a subset of papillomavirus 18-immortalized human prostate cells.

PubMed

Morgan, Kevin; Stavrou, Emmanouil; Leighton, Samuel P; Miller, Nicola; Sellar, Robin; Millar, Robert P

2011-06-15

Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated. Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis. High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists. Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists. Copyright © 2010 Wiley-Liss, Inc.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

PubMed

Zwart, Ruud; Reed, Hannah; Sher, Emanuele

2018-01-01

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabivarin

PubMed Central

Pertwee, R G

2007-01-01

Cannabis sativa is the source of a unique set of compounds known collectively as plant cannabinoids or phytocannabinoids. This review focuses on the manner with which three of these compounds, (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), (−)-cannabidiol (CBD) and (−)-trans-Δ9-tetrahydrocannabivarin (Δ9-THCV), interact with cannabinoid CB1 and CB2 receptors. Δ9-THC, the main psychotropic constituent of cannabis, is a CB1 and CB2 receptor partial agonist and in line with classical pharmacology, the responses it elicits appear to be strongly influenced both by the expression level and signalling efficiency of cannabinoid receptors and by ongoing endogenous cannabinoid release. CBD displays unexpectedly high potency as an antagonist of CB1/CB2 receptor agonists in CB1- and CB2-expressing cells or tissues, the manner with which it interacts with CB2 receptors providing a possible explanation for its ability to inhibit evoked immune cell migration. Δ9-THCV behaves as a potent CB2 receptor partial agonist in vitro. In contrast, it antagonizes cannabinoid receptor agonists in CB1-expressing tissues. This it does with relatively high potency and in a manner that is both tissue and ligand dependent. Δ9-THCV also interacts with CB1 receptors when administered in vivo, behaving either as a CB1 antagonist or, at higher doses, as a CB1 receptor agonist. Brief mention is also made in this review, first of the production by Δ9-THC of pharmacodynamic tolerance, second of current knowledge about the extent to which Δ9-THC, CBD and Δ9-THCV interact with pharmacological targets other than CB1 or CB2 receptors, and third of actual and potential therapeutic applications for each of these cannabinoids. PMID:17828291

Receptor specificity and trigemino-vascular inhibitory actions of a novel 5-HT1B/1D receptor partial agonist, 311C90 (zolmitriptan).

PubMed

Martin, G R; Robertson, A D; MacLennan, S J; Prentice, D J; Barrett, V J; Buckingham, J; Honey, A C; Giles, H; Moncada, S

1997-05-01

1. 311C90 (zolmitriptan zomig: (S)-4[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl]methyl]-2-oxazolidinone) is a novel 5-HT1B/1D receptor agonist with proven efficacy in the acute treatment of migraine. Here, we describe the receptor specificity of the drug and its actions on trigeminal-evoked plasma protein extravasation into the dura mater of the anaesthetized guinea-pig. 2. At the "5-HT1B-like' receptor mediating vascular contraction (rabbit saphenous vein), the compound was a potent (p[A50] = 6.79 +/- 0.06) partial agonist achieving 77 +/- 4% of the maximum effect to 5-hydroxytryptamine (5-HT). In the same experiments, sumatriptan (p[A50] = 6.48 +/- 0.04) was half as potent as 311C90 and produced 97 +/- 2% of the 5-HT maximum effect. Studies in which receptor inactivation methods were used to estimate the affinity (pKA) and efficacy relative to 5-HT (tau rel) for each agonist confirmed that 311C90 exhibits higher affinity than sumatriptan (pKA = 6.63 +/- 0.04 and 6.16 +/- 0.03, respectively) and that both drugs are partial agonists relative to 5-HT (tau rel = 0.61 +/- 0.03 and 0.63 +/- 0.10, respectively, compared to 5-HT = 1.0). 3. Consistent with its effects in rabbit saphenous vein, 311C90 also produced concentration-dependent contractions of primate basilar artery and human epicardial coronary artery rings. In basilar artery, agonist potency (p[A50] = 6.92 +/- 0.07) was similar to that demonstrated in rabbit saphenous vein, again being 2-3 fold higher than for sumatriptan (p[A50] = 6.46 +/- 0.03). Both agonists produced about 50% of the maximum response obtained with 5-HT in the same preparations. In rings of human coronary artery, the absolute potency of 311C90 and sumatriptan was higher than in primate basilar artery (p[A50] = 7.3 +/- 0.1 and 6.7 +/- 0.1, respectively), but maximum effects relative to 5-HT were lower (37 +/- 8% and 35 +/- 7%, respectively). In both types of vessel, the inability of 5-HT1B/1D agonists to achieve the same maximum as the endogenous agonist 5-HT is explained by the additional presence of 5-HT2A receptors. 4. 311C90 displayed high affinity at human recombinant 5-HT1D (formerly 5-HT1D alpha) and 5-HT1B (formerly 5-HT1D beta) receptors in transfected CHO-K1 cell membranes (pIC50 values = 9.16 +/- 0.12 and 8.32 +/- 0.09, respectively). In intact cells, the drug produced concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase (p[A50] = 9.9 and 9.5, respectively) achieving the same maximum effect as 5-HT. Excepting human recombinant 5-HT1A and 5-ht1F receptors at which the drug behaved as an agonist with modest affinity (pIC50 = 6.45 +/- 0.11 and 7.22 +/- 0.12, respectively), 311C90 exhibited low, or no detectable affinity (pKi or pKB < or = 5.5) at numerous other monoamine receptors, including other 5-HT receptor subtypes. 5. When administered to anaesthetized guinea-pigs ten minutes before unilateral electrical stimulation of the trigeminal ganglion (1.2 mA, 5 Hz, 5 ms, 5 min), 311C90 (3-30 micrograms kg-1, i.v.) caused a dose-dependent inhibition of [125I]-albumin extravasation within the ipsilateral dura mater. At the same doses, the drug also produced dose-dependent falls in cranial vascular conductance (32.3 +/- 7.5% at 30 micrograms kg-1), as measured in the ear by laser doppler flowmetry. 6. These results show that 311C90, a novel member of the 5-HT1B/1D agonist drug class, exhibits a high degree of pharmacological specificity. Its potent partial agonist action at "5-HT1B-like' receptors in intracranial arteries, coupled with potent agonism at 5-HT1D and 5-HT1B receptors and an ability to inhibit neurogenic plasma protein extravasation in the dura, are consistent with its utility as an effective acute treatment for migraine.

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

PubMed

Lefebvre, Romain A; Van Colen, Inge; Pauwelyn, Vicky; De Maeyer, Joris H

2016-06-15

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

PubMed Central

Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

2016-01-01

Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis. PMID:26947974

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed Central

Maggi, C. A.; Patacchini, R.; Eglezos, A.; Quartara, L.; Giuliani, S.; Giachetti, A.

1992-01-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1384907

Rate of Homologous Desensitization and Internalization of the GLP-1 Receptor.

PubMed

Shaaban, Ghina; Oriowo, Mabayoje; Al-Sabah, Suleiman

2016-12-26

The glucagon-like peptide-1 receptor (GLP-1R) is an important target in the treatment of type 2 diabetes mellitus. The aim of this study was to compare the rate of agonist stimulated desensitization and internalization of GLP-1R. To this end, an N-terminally myc-tagged GLP-1R was stably expressed in HEK-293 cells. Homologous desensitization was assessed by measuring the cAMP response to agonist stimulation following pre-incubation with agonist for up to 120 min. Receptor internalization was monitored using an indirect ELISA-based method and confocal microscopy. Pre-incubation with GLP-1 resulted in a time-dependent loss of response to a second stimulation. Washing cells following pre-incubation failed to bring cAMP levels back to basal. Taking this into account, two desensitization rates were calculated: "apparent" (t 1/2 = 19.27 min) and "net" (t 1/2 = 2.99 min). Incubation of cells with GLP-1 also resulted in a time-dependent loss of receptor cell surface expression (t 1/2 = 2.05 min). Rapid agonist-stimulated internalization of GLP-1R was confirmed using confocal microscopy. Stimulation of GLP-1R with GLP-1 results in rapid desensitization and internalization of the receptor. Interestingly, the rate of "net" desensitization closely matches the rate of internalization. Our results suggest that agonist-bound GLP-1R continues to generate cAMP after it has been internalized.

Putative antipsychotics with pronounced agonism at serotonin 5-HT1A and partial agonist activity at dopamine D2 receptors disrupt basal PPI of the startle reflex in rats.

PubMed

Auclair, Agnès L; Galinier, Alexandra; Besnard, Joël; Newman-Tancredi, Adrian; Depoortère, Ronan

2007-07-01

Prepulse inhibition (PPI) of the startle reflex has been extensively studied because it is disrupted in several psychiatric diseases, most notably schizophrenia. In rats, and to a lesser extent, in humans, PPI can be diminished by dopamine (DA) D(2)/D(3) and serotonin 5-HT(1A) receptor agonists. A novel class of potential antipsychotics (SSR181507, bifeprunox, and SLV313) possess partial agonist/antagonist properties at D(2) receptors and various levels of 5-HT(1A) activation. It thus appeared warranted to assess, in Sprague-Dawley rats, the effects of these antipsychotics on basal PPI. SSR181507, sarizotan, and bifeprunox decreased PPI, with a near-complete abolition at 2.5-10 mg/kg; SLV313 had a significant effect at 0.16 mg/kg only. Co-treatment with the 5-HT(1A) receptor antagonist WAY100,635 (0.63 mg/kg) showed that the 5-HT(1A) agonist activity of SSR181507 was responsible for its effect. By contrast, antipsychotics with low affinity and/or efficacy at 5-HT(1A) receptors, such as aripiprazole (another DA D(2)/D(3) and 5-HT(1A) ligand), and established typical and atypical antipsychotics (haloperidol, clozapine, risperidone, olanzapine, quetiapine, and ziprasidone) had no effect on basal PPI (0.01-2.5 to 2.5-40 mg/kg). The present data demonstrate that some putative antipsychotics with pronounced 5-HT(1A) agonist activity, coupled with partial agonist activity at DA D(2) receptors, markedly diminish PPI of the startle reflex in rats. These data raise the issue of the influence of such compounds on sensorimotor gating in humans.

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

PubMed

Pozvek, G; Hilton, J M; Quiza, M; Houssami, S; Sexton, P M

1997-04-01

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Human β-Defensin 1 and β-Defensin 3 (Mouse Ortholog mBD14) Function as Full Endogenous Agonists at Select Melanocortin Receptors.

PubMed

Ericson, Mark D; Singh, Anamika; Tala, Srinivasa R; Haslach, Erica M; Dirain, Marvin L S; Schaub, Jay W; Flores, Viktor; Eick, Natalie; Lensing, Cody J; Freeman, Katie T; Smeester, Branden A; Adank, Danielle N; Wilber, Stacey L; Speth, Robert; Haskell-Luevano, Carrie

2018-04-26

β-Defensin 3 (BD3) was identified as a ligand for the melanocortin receptors (MCRs) in 2007, although the pharmacology activity of BD3 has not been clearly elucidated. Herein, it is demonstrated that human BD3 and mouse BD3 are full micromolar agonists at the MCRs. Furthermore, mouse β-defensin 1 (BD1) and human BD1 are also MCR micromolar agonists. This work identifies BD1 as an endogenous MCR ligand and clarifies the controversial role of BD3 as a micromolar agonist.

Effects of kappa opioid agonists alone and in combination with cocaine on heart rate and blood pressure in conscious squirrel monkeys.

PubMed

Schindler, Charles W; Graczyk, Zofi; Gilman, Joanne P; Negus, S Stevens; Bergman, Jack; Mello, Nancy K; Goldberg, Steven R

2007-12-08

As kappa agonists have been proposed as treatments for cocaine abuse, the cardiovascular effects of the kappa opioid receptor agonists ethylketocyclazocine (EKC) and enadoline were investigated in conscious squirrel monkeys. Both EKC and enadoline increased heart rate with little effect on blood pressure. This effect appeared to be specific for kappa receptors as the mu opioid agonist morphine did not mimic the effects of the kappa agonists. The opioid antagonist naltrexone, at a dose of 1.0 mg/kg, blocked the effect of EKC. An action at both central and peripheral receptors may be responsible for the heart rate increase following kappa agonist treatment. The ganglionic blocker chlorisondamine partially antagonized the effect of EKC on heart rate, suggesting central involvement, while the peripherally-acting agonist ICI 204,448 ((+/-)-1-[2,3- (Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) also increased heart rate, supporting a peripheral site of action. When given in combination with cocaine, EKC produced effects that were sub-additive, suggesting that the kappa agonists may be used safely as cocaine abuse treatments.

Comparison of receptor mechanisms and efficacy requirements for delta-agonist-induced convulsive activity and antinociception in mice.

PubMed

Broom, Daniel C; Nitsche, Joshua F; Pintar, John E; Rice, Kenner C; Woods, James H; Traynor, John R

2002-11-01

Delta-opioid receptor-selective agonists produce antinociception and convulsions in several species, including mice. This article examines two hypotheses in mice: 1) that antinociception and convulsive activity are mediated through the same type of delta-receptor and 2) that greater delta-agonist efficacy is required for antinociception than for convulsive activity. Delta-mediated antinociception was evaluated in the acetic acid-induced abdominal constriction assay, which involves a low-intensity noxious stimulus; convulsive activity was indicated as a mild tonic-clonic convulsive episode followed by a period of catalepsy. In delta-opioid receptor knockout mice [DOR-1(-/-)], the nonpeptidic delta-agonists (+/-)-4-[(R*)-[(2S*,5R*)-2,5-dimethyl-4-(2-propenyl)-1- piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide hydrochloride (BW373U86) and (+)-4-[(R)-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N, N-diethylbenzamide (SNC80) failed to produce convulsive behavior demonstrating the absolute involvement of DOR-1 in this effect. In NIH Swiss mice expressing delta-opioid receptors, BW373U86 produced both antinociception and convulsive activity. These effects were antagonized by the putative delta(1)-receptor-selective antagonist 7-benzylidenenaltrexone and the putative delta(2)-receptor-selective antagonist naltriben. Tolerance developed to both the convulsive and antinociceptive effects of BW373U86. Tolerance to the convulsive, but not the antinociceptive, effects of BW373U86 was largely prevented when the antagonist naltrindole was given 20 min after each dose of the agonist in a 3-day treatment paradigm. The convulsive action of BW373U86 was also less sensitive than the antinociceptive action to treatment with the irreversible delta-antagonist naltrindole isothiocyanate. Collectively, these data suggest that the convulsive and antinociceptive activities of delta-agonists are mediated through the same receptor but that the receptor reserve for delta-mediated convulsive activity is greater than for delta-mediated antinociceptive activity.

Ischemic colitis related to sumatriptan overuse.

PubMed

Hodge, Joshua A; Hodge, Katherine D

2010-01-01

Serotonin-1 5-hydroxytryptamine (5-HT 1) receptor agonists are first line agents for migraine headaches. Patients with refractory headaches may use supratherapeutic doses of these medications. Described is a case of ischemic colitis related to overuse of sumatriptan. A 35-year-old woman presented with severe abdominal pain without diarrhea or hematochezia. For several days prior she had been self-treating a refractory migraine headache with frequent doses of sumatriptan. She is a nonsmoker and took no oral contraceptives or other serotonin agonists. A computed tomography scan of the abdomen revealed left-sided colitis. A colonoscopy with biopsy confirmed ischemic colitis and excluded inflammatory bowel disease (IBD). Previously published case reports have suggested an association between 5-HT 1 receptor agonists and ischemic colitis. These reports have been dismissed because the patients were taking oral contraceptives, serotonin agonists, or had other comorbidities. This healthy patient lacked risk factors for ischemia, is the youngest to be reported, and is the first without hematochezia. 5-HT 1 receptor agonists are generally considered safe. Ischemic colitis is a potentially serious complication of these agents. A retrospective review of 5-HT 1 receptor agonist users who have presented with acute onset abdominal pain or hematochezia is necessary to elucidate the incidence of this adverse event.

Dissociating anxiolytic and sedative effects of GABAAergic drugs using temperature and locomotor responses to acute stress

PubMed Central

Klanker, Marianne; Groenink, Lucianne; Korte, S. Mechiel; Cook, James M.; Van Linn, Michael L.; Hopkins, Seth C.; Olivier, Berend

2009-01-01

Rationale The stress-induced hyperthermia (SIH) model is an anxiety model that uses the transient rise in body temperature in response to acute stress. Benzodiazepines produce anxiolytic as well as sedative side effects through nonselective binding to GABAA receptor subunits. The GABAA receptor α1 subunit is associated with sedation, whereas the GABAA receptor α2 and α3 subunits are involved in anxiolytic effects. Objectives We therefore examined the effects of (non) subunit-selective GABAA receptor agonists on temperature and locomotor responses to novel cage stress. Results Using telemetric monitoring of temperature and locomotor activity, we found that nonsubunit-selective GABAA receptor agonist diazepam as well as the α3 subunit-selective receptor agonist TP003 dose-dependently attenuated SIH and locomotor responses. Administration of GABAA receptor α1-selective agonist zolpidem resulted in profound hypothermia and locomotor sedation. The GABAA receptor α1-selective antagonist βCCt antagonized the hypothermia, but did not reverse the SIH response attenuation caused by diazepam and zolpidem. These results suggest an important regulating role for the α1 subunit in thermoregulation and sedation. Ligands of extrasynaptic GABAA receptors such as alcohol and nonbenzodiazepine THIP attenuated the SIH response only at high doses. Conclusions The present study confirms a putative role for the GABAA receptor α1 subunit in hypothermia and sedation and supports a role for α2/3 subunit GABAA receptor agonists in anxiety processes. In conclusion, we show that home cage temperature and locomotor responses to novel home cage stress provide an excellent tool to assess both anxiolytic and sedative effects of various (subunit-selective) GABAAergic compounds. PMID:19169673

Antinociceptive effects of imidazoline I2 receptor agonists in the formalin test in rats

PubMed Central

Thorn, David A; Qiu, Yanyan; Zhang, Yanan; Li, Jun-Xu

2015-01-01

The imidazoline I2 receptor is an emerging drug target for analgesics. This study extended previous studies by examining the antinociceptive effects of three I2 receptor agonists (2-BFI, BU224 and CR4056) in the formalin test. The receptor mechanisms and anatomical mediation of I2 receptor agonist-induced antinociception were also examined. Formalin-induced flinching responses (2%, 50µl) were quantified after treatment with I2 receptor agonists alone or in combination with the I2 receptor antagonist idazoxan. Anatomical mediation was studied by locally administering 2-BFI into the plantar surface or into the right lateral ventricle via cannulae (i.c.v). The locomotor activity was also examined after central (i.c.v.) administration of 2-BFI. 2-BFI (1–10 mg/kg, i.p.) and BU224 (1–10 mg/kg, i.p.) attenuated the spontaneous flinching response observed during 10 min (phase 1) and 20–60 min (phase 2) following formalin treatment, while CR4056 (1–32 mg/kg, i.p.) only decreased phase 2 flinching response. The I2 receptor antagonist idazoxan attenuated the antinociceptive effects of 2-BFI and BU224 during phase 1, but not phase 2. Peripheral administration of 2-BFI (1–10 mg/kg, i.pl) to the hindpaw of rats had no antinociceptive effects. In contrast, centrally delivered 2-BFI (10–100 µg, i.c.v.) dose-dependently attenuated phase 1 and phase 2 flinching at doses that did not reduce the locomotor activity. Together, these data revealed the differential antinociceptive effects of I2 receptor agonists and the differential antagonism profiles by idazoxan, suggesting the involvement of different I2 receptor subtypes in reducing different phases of formalin-induced pain-like behaviors. In addition, the results also suggest the central mediation of I2 receptor agonist-induced antinociceptive actions. PMID:26599907

Facilitatory effects of selective agonists for tachykinin receptors on cholinergic neurotransmission: evidence for species differences.

PubMed Central

Belvisi, M. G.; Patacchini, R.; Barnes, P. J.; Maggi, C. A.

1994-01-01

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7516799

Agonistic autoantibodies against the angiotensin AT1 receptor increase in unstable angina patients after stent implantation.

PubMed

Tian, Miao; Sheng, Li; Huang, Peng; Li, Jun; Zhang, Chuan-Huan; Yang, Jun; Liao, Yu-Hua; Li, Liu-Dong

2014-12-01

Agonistic AT1 receptor autoantibodies have been described in patients with hypertension and preeclampsia. These autoantibodies could stimulate proliferation of vascular smooth muscle cells (VSMCs), which are involved in angiotensin II-induced vascular injury in cardiovascular disease. Hence, in this study, we explored the existence of agonistic AT1 receptor autoantibodies in unstable angina (UA) patients and the possible effects of them on the in-stent restenosis of these patients. A total of 95 UA patients and 98 healthy volunteers were enrolled. The serum of each patient was analyzed for the presence of AT1 receptor autoantibodies by enzyme-linked immunosorbent assay. Their effects on VSMC proliferation and c-fos and c-jun expression were studied in vitro. AT1 receptor autoantibodies were detected in 34/95 patients with UA. The incidence was 10.2% in the control group and rose to 47.37% after stent implantation. In vitro, this autoantibody had agonist-like activity, shown as stimulation of VSMC proliferation and upregulation of c-fos and c-jun expression. These effects were similar to that of angiotensin II and could be weakened partly by the AT1-receptor blocker valsartan. Our findings show that the autoantibody from UA patients has similar agonistic activity to angiotensin II and might play a role in the pathogenesis of in-stent restenosis in these patients.

Effects of histamine and 5-hydroxytryptamine on the growth rate of xenografted human bronchogenic carcinomas.

PubMed

Sheehan, P F; Baker, T; Tutton, P J; Barkla, D H

1996-01-01

1. The influence of histamine and 5-hydroxytryptamine (5-HT) antagonists and agonists on the volume doubling times (Td) of human bronchogenic carcinomas propagated as s.c. xenografts in immunosuppressed mice was examined. 2. The H2-receptor antagonists, cimetidine and ranitidine, increased Td. 3. Treatment with the H2-receptor agonist, 4-methyl histamine, had no effect on Td. 4. Co-administration of 4-methyl histamine and cimetidine abolished the effects of cimetidine. 5. The 5-HT2-receptor antagonists, cinanserin and ketanserin, both increased Td. 6. Treatment with the 5-HT1/2-receptor agonist quipazine (0.1 mg/kg, reflecting 5-HT2 agonist activity) decreased Td, while a higher dose (10.0 mg/kg) had no effect. 7. The 5-HT1/2-receptor antagonist, methiothepin, decreased Td. 8. The 5-HT uptake inhibitor, fluoxetine, increased Td in one tumour line but not in another, while the 5-HT releaser/depletor, fenfluramine, increased Td. 9. Histamine may stimulate tumour growth through the histamine H2-receptor, while the dominant effect of 5-HT is 5-HT1-receptor inhibition. 10. Tumour growth in some bronchogenic carcinomas may involve 5-HT uptake mechanisms.

Discovery of Tetrahydropyrazolopyridine as Sphingosine 1-Phosphate Receptor 3 (S1P3)-Sparing S1P1 Agonists Active at Low Oral Doses.

PubMed

Demont, Emmanuel H; Bailey, James M; Bit, Rino A; Brown, Jack A; Campbell, Colin A; Deeks, Nigel; Dowell, Simon J; Eldred, Colin; Gaskin, Pam; Gray, James R J; Haynes, Andrea; Hirst, David J; Holmes, Duncan S; Kumar, Umesh; Morse, Mary A; Osborne, Greg J; Renaux, Jessica F; Seal, Gail A L; Smethurst, Chris A; Taylor, Simon; Watson, Robert; Willis, Robert; Witherington, Jason

2016-02-11

FTY720 is the first oral small molecule approved for the treatment of people suffering from relapsing-remitting multiple sclerosis. It is a potent agonist of the S1P1 receptor, but its lack of selectivity against the S1P3 receptor has been linked to most of the cardiovascular side effects observed in the clinic. These findings have triggered intensive efforts toward the identification of a second generation of S1P3-sparing S1P1 agonists. We have recently disclosed a series of orally active tetrahydroisoquinoline (THIQ) compounds matching these criteria. In this paper we describe how we defined and implemented a strategy aiming at the discovery of selective structurally distinct follow-up agonists. This effort culminated with the identification of a series of orally active tetrahydropyrazolopyridines.

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid RaftsS⃞

PubMed Central

Qiu, Yu; Wang, Yan; Chen, Hong-Zhuan; Loh, Horace H.

2011-01-01

μ-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonist-induced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of β-arrestins. The translocation of β-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of β-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of β-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1. PMID:21518774

Oxytocin and Vasopressin Agonists and Antagonists as Research Tools and Potential Therapeutics

PubMed Central

Manning, M; Misicka, A; Olma, A; Bankowski, K; Stoev, S; Chini, B; Durroux, T; Mouillac, B; Corbani, M; Guillon, G

2012-01-01

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V1a, V1b and V2 receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V1b receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V1a agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V2/V1a antagonist, conivaptan and the V2 antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V1a, V1b and V2 antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences. PMID:22375852

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

PubMed Central

Beattie, D. T.; Stubbs, C. M.; Connor, H. E.; Feniuk, W.

1993-01-01

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter. PMID:7679026

Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells

PubMed Central

Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod; Coin, Irene

2017-01-01

The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors. DOI: http://dx.doi.org/10.7554/eLife.27711.001 PMID:28771403

Activation of 5-hydroxytryptamine1B/1D/1F receptors as a mechanism of action of antimigraine drugs.

PubMed

Ramírez Rosas, Martha B; Labruijere, Sieneke; Villalón, Carlos M; Maassen Vandenbrink, Antoinette

2013-08-01

The introduction of the triptans (5-hydroxytryptamine (5-HT)1B/1D receptor agonists) was a great improvement in the acute treatment of migraine. However, shortcomings of the triptans have prompted research on novel serotonergic targets for the treatment of migraine. In this review the different types of antimigraine drugs acting at 5-HT receptors, their discovery and development are discussed. The first specific antimigraine drugs were the ergot alkaloids, consisting of ergotamine, dihydroergotamine and methysergide, which are agonists at 5-HT receptors, but can also bind α-adrenoceptors and dopamine receptors. In the 1990s, the triptans became available on the market. They are 5-HT1B/1D receptor agonists, showing fewer side effects due to their receptor specificity. In the last years, compounds that bind specifically to 5-HT1D, 5-HT1F and 5-HT7 receptors have been explored for their antimigraine potential. Furthermore, the serotonergic system seems to act in tight connection with the glutamatergic as well as the CGRP-ergic systems, which may open novel therapeutic avenues. Although the triptans are very effective in treating migraine attacks, their shortcomings have stimulated the search for novel drugs. Currently, the focus is on 5-HT1F receptor agonists, which seem devoid of vascular side effects. Moreover, novel compounds that affect multiple transmitter and/or neuropeptide systems that are involved in migraine could be of therapeutic relevance.

Activation of lumbosacral 5-HT2C receptors induces bursts of rhythmic activity in sympathetic nerves to the vas deferens in male rats

PubMed Central

Stafford, Stuart A; Tang, Kim; Coote, John H

2006-01-01

We previously demonstrated that p-chloroamphetamine (PCA) intravenously (i.v.) evokes a specific patterned bursting response in the vas deferens nerve (VDN) of anaesthetised male rats that is associated with contraction of the vas deferens, and ejaculation and contraction of the bulbospongiosus muscles. The present study used selective 5-HT agonists to induce similar rhythmic bursting responses in the VDN in order to reveal the 5-HT receptor subtypes involved. The 5-HT2C receptor agonist (1.0 mg kg−1 Ro600175 i.v.) evoked the characteristic bursting pattern responses in the VDN. The 5-HT1A receptor agonist (1.0 mg kg−1 8-OH-DPAT i.v.) failed to elicit any responses. However, 8-OH-DPAT coadministered in combination with Ro600175 induced a potentiation of the responses. Responses were also evoked in rats with a mid-thoracic spinalisation, with a more predictable response being observed following the combination of agonists. This suggests an action of both agonists in the lumbosacral spinal cord. Responses were blocked by 0.5 mg kg−1 SB206553 i.v. (5-HT2B/C receptor antagonist) or 0.5 mg kg−1 WAY100635 i.v. (5-HT1A receptor antagonist), but not 0.1 or 1.0 mg kg−1 SB269970 i.v. (5-HT7 receptor antagonist). We suggest that activation of 5-HT2C and 5-HT1A receptor subtypes synergistically elicits contraction of the vas deferens through the activation of sympathetic preganglionic neurones in the spinal cord. These data support the idea of a proejaculatory action of 5-HT2C receptors in the lumbosacral spinal cord, suggesting a descending 5-HT excitatory pathway in addition to a 5-HT inhibitory pathway. An excitatory action of 8-OH-DPAT at lumbosacral sites is also evident. PMID:16799648

Transcriptional Modulation of the Immune Response by Peroxisome Proliferator-Activated Receptor-α Agonists in Autoimmune Disease1

PubMed Central

Gocke, Anne R.; Hussain, Rehana Z.; Yang, Yuhong; Peng, Haiyan; Weiner, Jeffrey; Ben, Li-Hong; Drew, Paul D.; Stuve, Olaf; Lovett-Racke, Amy E.; Racke, Michael K.

2010-01-01

Peroxisome proliferator-activated receptor-α (PPARα) agonists have been shown to have a therapeutic benefit in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). In this study, we investigated the mechanism by which the PPARα agonist gemfibrozil induces immune deviation and protects mice from EAE. We demonstrated that treatment with gemfibrozil increases expression of the Th2 transcription factor GATA-3 and decreases expression of the Th1 transcription factor T-bet in vitro and directly ex vivo. These changes correlated with an increase in nuclear PPARα expression. Moreover, the protective effects of PPARα agonists in EAE were shown to be partially dependent on IL-4 and to occur in a receptor-dependent manner. PPARα was demonstrated, for the first time, to regulate the IL-4 and IL-5 genes and to bind the IL-4 promoter in the presence of steroid receptor coactivator-1, indicating that PPARα can directly transactivate the IL-4 gene. Finally, therapeutic administration of PPARα agonists ameliorated clinically established EAE, suggesting that PPARα agonists may provide a treatment option for immune-mediated inflammatory diseases. PMID:19299749

Role of A1 and A2A adenosine receptor agonists in adipose tissue inflammation induced by obesity in mice.

PubMed

DeOliveira, Caroline Candida; Paiva Caria, Cintia Rabelo E; Ferreira Gotardo, Erica Martins; Ribeiro, Marcelo Lima; Gambero, Alessandra

2017-03-15

Adenosine receptors are expressed in adipose tissue and control physiological and pathological events such as lipolysis and inflammation. The aim of this study was to evaluate the activity of N 6 -cyclopentyladenosine (CPA), a potent and selective A 1 adenosine receptor agonist; 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine hydrochloride (CGS-21680), an A 2A adenosine receptor agonist; and 5'-N-ethylcarboxamidoadenosine (NECA), a potent non-selective adenosine receptor agonist on adipose tissue inflammatory alterations induced by obesity in mice. Swiss mice were fed with a high-fat diet for 12 weeks and agonists were administered in the last two weeks. Body weight, adiposity and glucose homeostasis were evaluated. Inflammation in adipose tissue was assessed by evaluation of adipokine production and macrophage infiltration. Adenosine receptor signaling in adipose tissue was also evaluated. Mice that received CGS21680 presented an improvement in glucose homeostasis in association with systemically reduced inflammatory markers (TNF-α, PAI-1) and in the visceral adipose tissue (TNF-α, MCP-1, macrophage infiltration). Activation of p38 signaling was found in adipose tissue of this group of mice. NECA-treated mice presented some improvements in glucose homeostasis associated with an observed weight loss. Mice that received CPA presented only a reduction in the ex vivo basal lipolysis rate measured within visceral adipose tissue. In conclusion, administration of the A 2A receptor agonist to obese mice resulted in improvements in glucose homeostasis and adipose tissue inflammation, corroborating the idea that new therapeutics to treat obesity could emerge from these compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

NOP Receptor Mediates Anti-analgesia Induced by Agonist-Antagonist Opioids

PubMed Central

Gear, Robert W.; Bogen, Oliver; Ferrari, Luiz F.; Green, Paul G.; Levine, Jon D.

2014-01-01

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ~90 minutes after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69,593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy Mu Opioid Receptor (MOR) Agonist/Delta Opioid Receptor (DOR) Antagonist Ligands

PubMed Central

Mosberg, Henry I.; Yeomans, Larisa; Harland, Aubrie A.; Bender, Aaron M.; Sobczyk-Kojiro, Katarzyna; Anand, Jessica P.; Clark, Mary J.; Jutkiewicz, Emily M.; Traynor, John R.

2013-01-01

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance. PMID:23419026

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function.

PubMed

Bower, Kiowa S; Price, Kerry L; Sturdee, Laura E C; Dayrell, Mariza; Dougherty, Dennis A; Lummis, Sarah C R

2008-02-12

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax/Imax 5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50=16 and 27 microM, and Ki for displacement of [3H]granisetron binding=0.8 and 1.8 microM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax=0.15), low affinity (EC50=113 microM; Ki=4.8 microM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50=8.1 microM; Ki=2.7 microM) but is a very weak partial agonist (Rmax=0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.

Development of 2′-substituted (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists

PubMed Central

Risgaard, Rune; Nielsen, Simon D.; Hansen, Kasper B.; Jensen, Christina M.; Nielsen, Birgitte; Traynelis, Stephen F.; Clausen, Rasmus P.

2013-01-01

A series of 2′-substituted analogues of the selective NMDA receptor ligand (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2′-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2′-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor. PMID:23614571

A Novel Non-Peptidic Agonist of the Ghrelin Receptor with Orexigenic Activity In vivo

NASA Astrophysics Data System (ADS)

Pastor-Cavada, Elena; Pardo, Leticia M.; Kandil, Dalia; Torres-Fuentes, Cristina; Clarke, Sarah L.; Shaban, Hamdy; McGlacken, Gerard P.; Schellekens, Harriet

2016-11-01

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand.

5-HT1A receptor blockade reverses GABAA receptor α3 subunit-mediated anxiolytic effects on stress-induced hyperthermia

PubMed Central

van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne

2010-01-01

Rationale Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABAA and the serotonin receptor system interact, a serotonergic component in the anxiolytic actions of benzodiazepines could be present. Objectives The main aim of the present study was to investigate whether the anxiolytic effects of (non-)selective α subunit GABAA receptor agonists could be reversed with 5-HT1A receptor blockade using the stress-induced hyperthermia (SIH) paradigm. Results The 5-HT1A receptor antagonist WAY-100635 (0.1–1 mg/kg) reversed the SIH-reducing effects of the non-α-subunit selective GABAA receptor agonist diazepam (1–4 mg/kg) and the GABAA receptor α3-subunit selective agonist TP003 (1 mg/kg), whereas WAY-100635 alone was without effect on the SIH response or basal body temperature. At the same time, co-administration of WAY-100635 with diazepam or TP003 reduced basal body temperature. WAY-100635 did not affect the SIH response when combined with the preferential α1-subunit GABAA receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature. Conclusions The present study suggests an interaction between GABAA receptor α-subunits and 5-HT1A receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABAA receptor α3-subunits. Further understanding of the interactions between the GABAA and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs. PMID:20535452

Forskolin-free cAMP assay for Gi-coupled receptors.

PubMed

Gilissen, Julie; Geubelle, Pierre; Dupuis, Nadine; Laschet, Céline; Pirotte, Bernard; Hanson, Julien

2015-12-01

G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands. Copyright © 2015 Elsevier Inc. All rights reserved.

Glutamate metabotropic receptors as targets for drug therapy in epilepsy.

PubMed

Moldrich, Randal X; Chapman, Astrid G; De Sarro, Giovambattista; Meldrum, Brian S

2003-08-22

Metabotropic glutamate (mGlu) receptors have multiple actions on neuronal excitability through G-protein-linked modifications of enzymes and ion channels. They act presynaptically to modify glutamatergic and gamma-aminobutyric acid (GABA)-ergic transmission and can contribute to long-term changes in synaptic function. The recent identification of subtype-selective agonists and antagonists has permitted evaluation of mGlu receptors as potential targets in the treatment of epilepsy. Agonists acting on group I mGlu receptors (mGlu1 and mGlu5) are convulsant. Antagonists acting on mGlu1 or mGlu5 receptors are anticonvulsant against 3,5-dihydroxyphenylglycine (DHPG)-induced seizures and in mouse models of generalized motor seizures and absence seizures. The competitive, phenylglycine mGlu1/5 receptor antagonists generally require intracerebroventricular administration for potent anticonvulsant efficacy but noncompetitive antagonists, e.g., (3aS,6aS)-6a-naphthalen-2-ylmethyl-5-methyliden-hexahydrocyclopenta[c]furan-1-on (BAY36-7620), 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) block generalized seizures with systemic administration. Agonists acting on group II mGlu receptors (mGlu2, mGlu3) to reduce glutamate release are anticonvulsant, e.g., 2R,4R-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC], (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268). The classical agonists acting on group III mGlu receptors such as L-(+)-2-amino-4-phosphonobutyric acid, and L-serine-O-phosphate are acutely proconvulsant with some anticonvulsant activity. The more recently identified agonists (R,S)-4-phosphonophenylglycine [(R,S)-PPG] and (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG] and (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid [ACPT-1] are all anticonvulsant without proconvulsant effects. Studies in animal models of kindling reveal some efficacy of mGlu receptor ligands against fully kindled limbic seizures. In genetic mouse models, mGlu1/5 antagonists and mGlu2/3 agonists are effective against absence seizures. Thus, antagonists at group I mGlu receptors and agonists at groups II and III mGlu receptors are potential antiepileptic agents, but their clinical usefulness will depend on their acute and chronic side effects. Potential also exists for combining mGlu receptor ligands with other glutamatergic and non-glutamatergic agents to produce an enhanced anticonvulsant effect. This review also discusses what is known about mGlu receptor expression and function in rodent epilepsy models and human epileptic conditions.

(+)Lysergic acid diethylamide, but not its nonhallucinogenic congeners, is a potent serotonin 5HT1C receptor agonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burris, K.D.; Breeding, M.; Sanders-Bush, E.

Activation of central serotonin 5HT2 receptors is believed to be the primary mechanism whereby lysergic acid diethylamide (LSD) and other hallucinogens induce psychoactive effects. This hypothesis is based on extensive radioligand binding and electrophysiological and behavioral studies in laboratory animals. However, the pharmacological profiles of 5HT2 and 5HT1C receptors are similar, making it difficult to distinguish between effects due to activation of one or the other receptor. For this reason, it was of interest to investigate the interaction of LSD with 5HT1C receptors. Agonist-stimulated phosphoinositide hydrolysis in rat choroid plexus was used as a direct measure of 5HT1C receptor activation.more » (+)LSD potently stimulated phosphoinositide hydrolysis in intact choroid plexus and in cultures of choroid plexus epithelial cells, with EC50 values of 9 and 26 nM, respectively. The effect of (+)LSD in both systems was blocked by 5HT receptor antagonists with an order of activity consistent with interaction at 5HT1C receptors. Neither (+)-2-bromo-LSD nor lisuride, two nonhallucinogenic congeners of LSD, were able to stimulate 5HT1C receptors in cultured cells or intact choroid plexus. In contrast, lisuride, like (+)LSD, is a partial agonist at 5HT2 receptors in cerebral cortex slices and in NIH 3T3 cells transfected with 5HT2 receptor cDNA. The present finding that (+)LSD, but not its nonhallucinogenic congeners, is a 5HT1C receptor agonist suggests a possible role for these receptors in mediating the psychoactive effects of LSD.« less

Dopamine D2-like receptor signaling suppresses human osteoclastogenesis.

PubMed

Hanami, Kentaro; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Yamaoka, Kunihiro; Kubo, Satoshi; Kondo, Masahiro; Tanaka, Yoshiya

2013-09-01

Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. Although the relevance of neuroendocrine system to bone metabolism has been emerging, the precise effects of dopaminergic signaling upon osteoclastogenesis remain unknown. Here, we demonstrate that human monocyte-derived osteoclast precursor cells express all dopamine-receptor subtypes. Dopamine and dopamine D2-like receptor agonists such as pramipexole and quinpirole reduced the formation of TRAP-positive multi-nucleated cells, cathepsin K mRNA expression, and pit formation area in vitro. These inhibitory effects were reversed by pre-treatment with a D2-like receptor antagonist haloperidol or a Gαi inhibitor pertussis toxin, but not with the D1-like receptor antagonist SCH-23390. Dopamine and dopamine D2-like receptor agonists, but not a D1-like receptor agonist, suppressed intracellular cAMP concentration as well as RANKL-meditated induction of c-Fos and NFATc1 mRNA expression in human osteoclast precursor cells. Finally, the dopamine D2-like receptor agonist suppressed LPS-induced osteoclast formation in murine bone marrow culture ex vivo. These findings indicate that dopaminergic signaling plays an important role in bone homeostasis via direct effects upon osteoclast differentiation and further suggest that the clinical use of neuroleptics is likely to affect bone mass. Copyright © 2013 Elsevier Inc. All rights reserved.

Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test.

PubMed

Rizzi, A; Ruzza, C; Bianco, S; Trapella, C; Calo', G

2017-08-01

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1-1mgkg -1 ) and morphine (0.1-10mgkg -1 ) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01-0.1mgkg -1 ) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of central histamine receptors blockade on GABA(A) agonist-induced food intake in broiler cockerels.

PubMed

Morteza, Zendehdel; Vahhab, Babapour; Hossein, Jonaidi

2008-02-01

In this study, the effect of intracerebroventricular (i.c.v) injection of H1, H2 and H3 antagonists on feed intake induced by GABA(A) agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABA(A) agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p < or = 0.05), significantly, while the famotidine and thioperamide were ineffective. These results suggest the existence of H1-receptor mediated histamine-GABA(A) receptor interaction on food intake in broiler cockerels.

Synthesis and pharmacological profile of a new selective G protein-coupled receptor 119 agonist; 6-((2-fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one.

PubMed

Sakairi, Masao; Kogami, Masakazu; Torii, Masafumi; Makino, Mitsuhiro; Kataoka, Daisuke; Okamoto, Ryuji; Miyazawa, Toshiyuki; Inoue, Megumi; Takahashi, Naoki; Harada, Satoko; Watanabe, Nobuhide

2012-01-01

6-((2-Fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one is a potent drug-like G protein-coupled receptor 119 (GPR119) agonist. It is hoped that this compound would be instrumental in probing the pharmacological potential of GPR119 agonists.

Involvement of adenosine A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats.

PubMed

Justinova, Zuzana; Ferre, Sergi; Segal, Pavan N; Antoniou, Katerina; Solinas, Marcello; Pappas, Lara A; Highkin, Jena L; Hockemeyer, Jorg; Munzar, Patrik; Goldberg, Steven R

2003-12-01

Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the discriminative-stimulus effects of methamphetamine and cocaine.

Sarizotan, a serotonin 5-HT1A receptor agonist and dopamine receptor ligand. 1. Neurochemical profile.

PubMed

Bartoszyk, G D; Van Amsterdam, C; Greiner, H E; Rautenberg, W; Russ, H; Seyfried, C A

2004-02-01

Sarizotan exhibited high affinities only to serotonin 5-HT1A receptors and dopamine DA D4>D3>D2 receptors with the profile of a 5-HT1A agonist and DA antagonist demonstrated by the inhibition of cAMP-stimulation and guinea pig ileum contraction, decreased accumulation of the 5-HT precursor 5-hydroxytryptophan and increased levels of 5-HT metabolites, increased accumulation of DA precursor dihydroxyphenylalanine (DOPA) and the reduced levels of DA metabolites in intact rats. However, sarizotan at higher doses decreased DA precursor accumulation in reserpinized rats and induced contralateral rotational behavior in unilaterally substantia nigra lesioned rats, indicating some intrinsic dopaminergic activity; at D2 receptors sarizotan may act as a partial agonist, depending on the dopaminergic impulse flow. Sarizotan represents a new approach for the treatment of extrapyramidal motor complications such as l-DOPA-induced dyskinesia in Parkinson's disease.

Fixed ratio combinations of glucagon like peptide 1 receptor agonists with basal insulin: a systematic review and meta-analysis.

PubMed

Liakopoulou, Paraskevi; Liakos, Aris; Vasilakou, Despoina; Athanasiadou, Eleni; Bekiari, Eleni; Kazakos, Kyriakos; Tsapas, Apostolos

2017-06-01

Basal insulin controls primarily fasting plasma glucose but causes hypoglycaemia and weight gain, whilst glucagon like peptide 1 receptor agonists induce weight loss without increasing risk for hypoglycaemia. We conducted a systematic review and meta-analysis of randomised controlled trials to investigate the efficacy and safety of fixed ratio combinations of basal insulin with glucagon like peptide 1 receptor agonists. We searched Medline, Embase, and the Cochrane Library as well as conference abstracts up to December 2016. We assessed change in haemoglobin A 1c , body weight, and incidence of hypoglycaemia and gastrointestinal adverse events. We included eight studies with 5732 participants in the systematic review. Switch from basal insulin to fixed ratio combinations with a glucagon like peptide 1 receptor agonist was associated with 0.72% reduction in haemoglobin A 1c [95% confidence interval -1.03 to -0.41; I 2  = 93%] and 2.35 kg reduction in body weight (95% confidence interval -3.52 to -1.19; I 2  = 93%), reducing also risk for hypoglycaemia [odds ratio 0.70; 95% confidence interval 0.57 to 0.86; I 2  = 85%] but increasing incidence of nausea (odds ratio 6.89; 95% confidence interval 3.73-12.74; I 2  = 79%). Similarly, switching patients from treatment with a glucagon like peptide 1 receptor agonist to a fixed ratio combination with basal insulin was associated with 0.94% reduction in haemoglobin A 1c (95% confidence interval -1.11 to -0.77) and an increase in body weight by 2.89 kg (95% confidence interval 2.17-3.61). Fixed ratio combinations of basal insulin with glucagon like peptide 1 receptor agonists improve glycaemic control whilst balancing out risk for hypoglycaemia and gastrointestinal side effects.

Lorcaserin, a novel selective human 5-hydroxytryptamine2C agonist: in vitro and in vivo pharmacological characterization.

PubMed

Thomsen, William J; Grottick, Andrew J; Menzaghi, Frederique; Reyes-Saldana, Hazel; Espitia, Stephen; Yuskin, Diane; Whelan, Kevin; Martin, Michael; Morgan, Michael; Chen, Weichao; Al-Shamma, Hussien; Smith, Brian; Chalmers, Derek; Behan, Dominic

2008-05-01

5-Hydroxytryptamine (5-HT)(2C) receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT(2C) full agonist. Lorcaserin bound to human and rat 5-HT(2C) receptors with high affinity (K(i) = 15 +/- 1 nM, 29 +/- 7 nM, respectively), and it was a full agonist for the human 5-HT(2C) receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT(2A) and 5-HT(2B) receptors, respectively. Lorcaserin was also highly selective for human 5-HT(2C) over other human 5-HT receptors (5-HT(1A), 5-HT(3), 5-HT(4C), 5-HT5(5A), 5-HT(6), and 5-HT(7)), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT(2A) agonist (+/-)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT(2A) agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT(2C)-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT(2A) antagonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT(2C) receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT(2C) receptor, with potential for the treatment of obesity.

Structure-Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma.

PubMed

Franchini, Silvia; Sorbi, Claudia; Battisti, Umberto Maria; Tait, Annalisa; Bencheva, Leda Ivanova; Cichero, Elena; Fossa, Paola; Cilia, Antonio; Prezzavento, Orazio; Ronsisvalle, Simone; Aricò, Giuseppina; Benassi, Luisa; Vaschieri, Cristina; Azzoni, Paola; Magnoni, Cristina; Brasili, Livio

2017-11-22

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ 1 R; three compounds were shown to be σ 1 R agonists, while another proved to be the only σ 1 R antagonist. Only one of the σ 1 R agonists (BS148) also exhibited σ 2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ 2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

NASA Astrophysics Data System (ADS)

di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

2015-11-01

Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

PubMed

Smith, R L; Canton, H; Barrett, R J; Sanders-Bush, E

1998-11-01

Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y1 Receptor Agonists

PubMed Central

Ravi, R. Gnana; Kim, Hak Sung; Servos, Jörg; Zimmermann, Herbert; Lee, Kyeong; Maddileti, Savitri; Boyer, José L.; Harden, T. Kendall; Jacobson, Kenneth A.

2016-01-01

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y1, P2Y2, P2Y4, and P2Y11 receptors, but not P2Y6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208–218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y1 or human P2Y1 and P2Y2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC50 at the hP2Y1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl–(N)-methanocarba-ATP and its N6-Me analogue were also highly selective, full agonists at P2Y1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y1 receptor agonist with an EC50 of 160 nM and was selective versus hP2Y2 and hP2Y4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y1 receptors. PMID:11985476

UTILIZATION OF THE LEAST SHREW AS A RAPID AND SELECTIVE SCREENING MODEL FOR THE ANTIEMETIC POTENTIAL AND BRAIN PENETRATION OF SUBSTANCE P AND NK1 RECEPTOR ANTAGONISTS

PubMed Central

Darmani, Nissar A.; Wang, Yaozhi; Abad, Joseph; Ray, Andrew P.; Thrush, Gerald R.; Ramirez, Juan

2008-01-01

Substance P (SP) is thought to play a cardinal role in emesis via the activation of central tachykinin NK1 receptors during the delayed phase of vomiting produced by chemotherapeutics. Although the existing supportive evidence is significant, due to lack of an appropriate animal model, the evidence is indirect. As yet, no study has confirmed that emesis produced by SP or a selective NK1 receptor agonist is sensitive to brain penetrating antagonists of either NK1, NK2, or NK3 receptors. The goals of this investigation were to demonstrate: 1) whether intraperitoneal (i.p.) administration of either SP, a brain penetrating (GR73632) or non-penetrating (e.g. SarMet – SP) NK1 receptor agonist, an NK2 receptor agonist (GR64349), or an NK3 receptor agonist (Pro7-NKB), would induce vomiting and/or scratching in the least shrew (Cryptotis parva) in a dose-dependent manner; and whether these effects are sensitive to the above selective receptor antagonists; 2) whether an exogenous emetic dose of SP (50 mg/kg, i.p.) can penetrate into the shrew brain stem and frontal cortex; 3) whether GR73632 (2.5 mg/kg, i.p.)-induced activation of NK1 receptors increases Fos-measured neuronal activity in the neurons of both brain stem emetic nuclei and the enteric nervous system of the gut; and 4) whether selective ablation of peripheral NK1 receptors can affect emesis produced by GR73632. The results clearly demonstrated that while SP produced vomiting only, GR73632 caused both emesis and scratching behavior dose-dependently in shrews, and these effects were sensitive to NK1-, but not NK2- or NK3-receptor antagonists. Neither the selective, non-penetrating NK1 receptor agonists, nor the selective NK2- or NK3-receptor agonists, caused a significant dose-dependent behavioral effect. An emetic dose of SP selectively and rapidly penetrated the brain stem but not the frontal cortex. Systemic GR73632 increased Fos expression in the enteric nerve plexi, the medial subnucleus of nucleus tractus solitarius, and the dorsal motor nucleus of the vagus, but not the area postrema. Ablation of peripheral NK1 receptors attenuated the ability of GR73632 to induce a maximal frequency of emesis and shifted its percent animals vomiting dose-response curve to the right. The NK1-ablated shrews exhibited scratching behavior after systemic GR73632-injection. These results, for the first time, affirm a cardinal role for central NK1 receptors in SP-induced vomiting, and a facilitatory role for gastrointestinal NK1 receptors. In addition, these data support the validation of the least shrew as a specific and rapid behavioral animal model to screen concomitantly both the CNS penetration and the antiemetic potential of tachykinin NK1 receptor antagonists. PMID:18471804

In vivo dopamine agonist properties of rotigotine: Role of D1 and D2 receptors.

PubMed

Fenu, Sandro; Espa, Elena; Pisanu, Augusta; Di Chiara, Gaetano

2016-10-05

Rotigotine acts in vitro as a full agonist of dopamine D1 receptors at concentrations almost superimposable to those at which it acts on D2 receptors. However in vivo evidence of the differences between the agonist activity of rotigotine at D1 receptors from that on the D2 receptors has not been provided yet. In order to test the ability of rotigotine to stimulate dopamine D1 and D2 receptors in vivo, we studied the effect of SCH39166 and eticlopride, selective dopamine D1 and D2/D3 receptor antagonists respectively, on rotigotine-induced contralateral turning behavior in 6-hydroxydopamine lesioned rats. Furthermore, the expression of the immediate-early gene c-fos in the caudate-putamen, was evaluated. As a comparison, we tested the D2/D3 agonist pramipexole. In primed rats, rotigotine (0.035, 0.1 and 0.35mg/kg) induced dose-dependent contralateral turning. Turning induced by 0.1mg/kg of rotigotine was reduced by pretreatment with the D1 antagonist SCH39166 and the D2 antagonist eticlopride. In drug-naive rats, rotigotine was less effective in eliciting turning but SCH39166 still reduced turning induced by rotigotine (0.35mg/kg). Pramipexole induced contralateral turning only in primed rats. SCH39166 potentiated and eticlopride abolished pramipexole-induced turning. Rotigotine induced Fos expression in the caudate-putamen and SCH39166 completely blocked it. Pramipexole failed to induce Fos. These results indicate that rotigotine acts in vivo as an agonist of D1 and D2 receptors while pramipexole is devoid of D1 activity in vivo. Given their differing DA receptor profiles, rotigotine and pramipexole might differ in their spectrum of application to the therapy of Parkinson's disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Xenopus-derived glucagon-like peptide-1 and polyethylene-glycosylated glucagon-like peptide-1 receptor agonists: long-acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities.

PubMed

Han, Jing; Fei, Yingying; Zhou, Feng; Chen, Xinyu; Zhang, Ying; Liu, Lin; Fu, Junjie

2018-02-01

Incretin-based therapies based on glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments of type 2 diabetes. Abundant research has focused on the development of long-acting GLP-1 receptor agonists. However, all GLP-1 receptor agonists in clinical use or development are based on human or Gila GLP-1. We have identified a potent GLP-1 receptor agonist, xGLP-1B, based on Xenopus GLP-1. To further modify the structure of xGLP-1B, alanine scanning was performed to study the structure -activity relationship of xGLP-1B. Two strategies were then employed to improve bioactivity. First, the C-terminal tail of lixisenatide was appended to cysteine-altered xGLP-1B analogues. Second, polyethylene glycol (PEG) chains with different molecular weights were conjugated with the peptides, giving a series of PEGylated conjugates. Comprehensive bioactivity studies of these conjugates were performed in vitro and in vivo. From the in vitro receptor activation potency and in vivo acute hypoglycaemic activities of conjugates 25 -36, 33 was identified as the best candidate for further biological assessments. Conjugate 33 exhibited prominent hypoglycaemic and insulinotropic activities, as well as improved pharmacokinetic profiles in vivo. The prolonged antidiabetic duration of 33 was further confirmed by pre-oral glucose tolerance tests (OGTT) and multiple OGTT. Furthermore, chronic treatment of db/db mice with 33 ameliorated non-fasting blood glucose and insulin levels, reduced HbA1c values and normalized their impaired glucose tolerance. Importantly, no in vivo toxicity was observed in mice treated with 33. Peptide 33 is a promising long-acting type 2 diabetes therapeutic deserving further investigation. © 2017 The British Pharmacological Society.

β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

PubMed Central

Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

2014-01-01

Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

5-HT6 receptor agonists and antagonists enhance learning and memory in a conditioned emotion response paradigm by modulation of cholinergic and glutamatergic mechanisms

PubMed Central

Woods, S; Clarke, NN; Layfield, R; Fone, KCF

2012-01-01

BACKGROUND AND PURPOSE 5-HT6 receptors are abundant in the hippocampus, nucleus accumbens and striatum, supporting their role in learning and memory. Selective 5-HT6 receptor antagonists produce pro-cognitive effects in several learning and memory paradigms while 5-HT6 receptor agonists have been found to enhance and impair memory. EXPERIMENTAL APPROACH The conditioned emotion response (CER) paradigm was validated in rats. Then we examined the effect of the 5-HT6 receptor antagonist, EMD 386088 (10 mg·kg−1, i.p.), and agonists, E-6801 (2.5 mg·kg−1, i.p.) and EMD 386088 (5 mg·kg−1, i.p.) on CER-induced behaviour either alone or after induction of memory impairment by the muscarinic receptor antagonist, scopolamine (0.3 mg·kg−1, i.p) or the NMDA receptor antagonist, MK-801 (0.1 mg·kg−1, i.p). KEY RESULTS Pairing unavoidable foot shocks with a light and tone cue during CER training induced a robust freezing response, providing a quantitative index of contextual memory when the rat was returned to the shock chamber 24 h later. Pretreatment (−20 min pre-training) with scopolamine or MK-801 reduced contextual freezing 24 h after CER training, showing production of memory impairment. Immediate post-training administration of 5-HT6 receptor antagonist, SB-270146, and agonists, EMD 386088 and E-6801, had little effect on CER freezing when given alone, but all significantly reversed scopolamine- and MK-801-induced reduction in freezing. CONCLUSION AND IMPLICATIONS Both the 5-HT6 receptor agonists and antagonist reversed cholinergic- and glutamatergic-induced deficits in associative learning. These findings support the therapeutic potential of 5-HT6 receptor compounds in the treatment of cognitive dysfunction, such as seen in Alzheimer's disease and schizophrenia. PMID:22568655

Characterizing novel metabolic pathways of melatonin receptor agonist agomelatine using metabolomic approaches

USDA-ARS?s Scientific Manuscript database

Agomelatine (AGM), an analog of melatonin, is a potential agonist at melatonin receptors 1/2 and a selective antagonist at 5-hydroxytryptamine 2C receptors. AGM is widely used for the treatment of major depressive episodes in adults. However, multiple adverse effects associated with AGM have been re...

Endogenous cannabinoid receptor agonists inhibit neurogenic inflammations in guinea pig airways.

PubMed

Yoshihara, Shigemi; Morimoto, Hiroshi; Ohori, Makoto; Yamada, Yumi; Abe, Toshio; Arisaka, Osamu

2005-09-01

Although neurogenic inflammation via the activation of C fibers in the airway must have an important role in the pathogenesis of asthma, their regulatory mechanism remains uncertain. The pharmacological profiles of endogenous cannabinoid receptor agonists on the activation of C fibers in airway tissues were investigated and the mechanisms how cannabinoids regulate airway inflammatory reactions were clarified. The effects of endogenous cannabinoid receptor agonists on electrical field stimulation-induced bronchial smooth muscle contraction, capsaicin-induced bronchoconstriction and capsaicin-induced substance P release in guinea pig airway tissues were investigated. The influences of cannabinoid receptor antagonists and K+ channel blockers to the effects of cannabinoid receptor agonists on these respiratory reactions were examined. Both endogenous cannabinoid receptor agonists, anandamide and palmitoylethanolamide, inhibited electrical field stimulation-induced guinea pig bronchial smooth muscle contraction, but not neurokinin A-induced contraction. A cannabinoid CB2 antagonist, SR 144528, reduced the inhibitory effect of endogenous agonists, but not a cannabinoid CB1 antagonist, SR 141716A. Inhibitory effects of agonists were also reduced by the pretreatment of large conductance Ca2+ -activated K+ channel (maxi-K+ channel) blockers, iberiotoxin and charybdotoxin, but not by other K+ channel blockers, dendrotoxin or glibenclamide. Anandamide and palmitoylethanolamide blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea pig airway tissues. Additionally, intravenous injection of palmitoylethanolamide dose-dependently inhibited capsaicin-induced guinea pig bronchoconstriction, but not neurokinin A-induced reaction. However, anandamide did not reduce capsaicin-induced guinea pig bronchoconstriction. These findings suggest that endogenous cannabinoid receptor agonists inhibit the activation of C fibers via cannabinoid CB2 receptors and maxi-K+ channels in guinea pig airways. Copyright (c) 2005 S. Karger AG, Basel.

Neonatal (+)-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists

PubMed Central

Graham, Devon L.; Amos-Kroohs, Robyn M.; Braun, Amanda A.; Grace, Curtis E.; Schaefer, Tori L.; Skelton, Matthew R.; Williams, Michael T.; Vorhees, Charles V.

2015-01-01

Neonatal exposure to (+)-methamphetamine (Meth) results in long-term behavioural abnormalities but its developmental mechanisms are unknown. In a series of experiments, rats were treated from post-natal days (PD) 11–20 (stage that approximates human development from the second to third trimester) with Meth or saline and assessed using locomotor activity as the readout following pharmacological challenge doses with dopamine, serotonin and glutamate agonists or antagonists during adulthood. Exposure to Meth early in life resulted in an exaggerated adult locomotor hyperactivity response to the dopamine D1 agonist SKF-82958 at multiple doses, a high dose only under-response activating effect of the D2 agonist quinpirole, and an exaggerated under-response to the activating effect of the N-methyl-D-aspartic acid (NMDA) receptor antagonist, MK-801. No change in locomotor response was seen following challenge with the 5-HT releaser p-chloroamphetamine or the 5-HT2/3 receptor agonist, quipazine. These are the first data to show that PD 11-20 Meth exposure induces long-lasting alterations to dopamine D1, D2 and glutamate NMDA receptor function and may suggest how developmental Meth exposure leads to many of its long-term adverse effects. PMID:22391043

Toll-like receptor agonists are potent inhibitors of human immunodeficiency virus-type 1 replication in peripheral blood mononuclear cells.

PubMed

Buitendijk, Maarten; Eszterhas, Susan K; Howell, Alexandra L

2014-05-01

Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). When agonists to TLR3, TLR7, TLR8 and TLR9 were added prior to infection with HIV-1, they significantly reduced infection of peripheral blood mononuclear cells. Interestingly, agonists to TLR8 and TLR9 were highly effective at blocking HIV replication even when added as late as 48 h or 72 h, respectively, after HIV-1 infection, indicating that the anti-viral effect was durable and long lasting. Analysis of the induction of anti-viral genes after agonist activation of TLR indicated that all of the agonists induced expression of the type I interferons and interferon stimulated genes, although to variable levels that depended on the agonist used. Interestingly, only the agonist to TLR9, ODN2395 DNA, induced expression of type II interferon and the anti-HIV proteins Apobec3G and SAMHD1. By blocking TLR activity using an inhibitor to the MyD88 adaptor protein, we demonstrated that, at least for TLR8 and TLR9, the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in HIV target cells. These findings suggest that agonists to the endosomal TLR function to induce expression of anti-HIV molecules by both TLR-mediated and non-TLR-mediated mechanisms. Moreover, the non-TLR-mediated mechanisms induced by these agonists could potentially be exploited to block HIV-1 replication in recently HIV-exposed individuals.

Antipsychotics differ in their ability to internalise human dopamine D2S and human serotonin 5-HT1A receptors in HEK293 cells.

PubMed

Heusler, Peter; Newman-Tancredi, Adrian; Loock, Timothé; Cussac, Didier

2008-02-26

Antipsychotic drugs act preferentially via dopamine D(2) receptor blockade, but interaction with serotonin 5-HT(1A) receptors has attracted interest as additional target for antipsychotic treatment. As receptor internalisation is considered crucial for drug action, we tested the propensity of antipsychotics to internalise human (h)D(2S) receptors and h5-HT(1A) receptors. Agonist-induced internalisation of hemaglutinin (HA)-tagged hD(2S) and HA-h5-HT(1A) receptors expressed in HEK293 cells was increased by coexpression of G-protein coupled receptor kinase 2 and beta-arrestin2. At the HA-hD(2S) receptor, dopamine, quinpirole and bromocriptine behaved as full agonists, while S(-)-3-(3-hydroxyphenyl)-N-n-propylpiperidine [(-)-3PPP] and sarizotan were partial agonists. The typical antipsychotic, haloperidol, and the atypical compounds, olanzapine, nemonapride, ziprasidone and clozapine did not internalise HA-hD(2S) receptors, whereas aripiprazole potently internalised these receptors (>50% relative efficacy). Among antipsychotics with combined D(2)/5-HT(1A) properties, bifeprunox and (3-exo)-8-benzoyl-N-[[(2S)7-chloro-2,3-dihydro-1,4-benzodioxin-1-yl]methyl]-8-azabicyclo-[3.2.1]octane-3-methanamine (SSR181507) partially internalised HA-hD(2S) receptors, piperazine, 1-(2,3-dihydro-1,4-benzodioxin-5-yl)-4-[[5-(4-fluorophenyl)-3-pyridinyl]methyl (SLV313) and N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine (F15063) were inactive. At the HA-h5-HT(1A) receptor, serotonin, (+)-8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] and sarizotan were full agonists, buspirone acted as partial agonist. (-)-Pindolol showed little activity and no internalising properties were manifested for the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635). Most antipsychotics induced HA-h5-HT(1A) receptor internalisation, with an efficacy rank order: nemonapride>F15063>SSR181507>bifeprunox approximately SLV313 approximately ziprasidone>aripiprazole and potencies: SLV313>SSR181507 approximately F15063>bifeprunox approximately nemonapride approximately aripiprazole>ziprasidone. Interestingly, the internalisation induced by clozapine was only minimal, whereas aripirazole and bifeprunox were more potent for internalisation than for G-protein activation. These different profiles of antipsychotics for receptor internalisation may help to evaluate their potential therapeutic impact in the treatment of schizophrenia.

Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

PubMed

Newman-Tancredi, A; Verrièle, L; Touzard, M; Millan, M J

2001-10-05

5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: Heterologous regulation of the same K+ channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamatchi, G.L.; Ticku, M.K.

1991-02-01

The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between themmore » when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.« less

Further evidence that tachykinin-induced contraction of human isolated bronchus is mediated only by NK2-receptors.

PubMed

Sheldrick, R L; Rabe, K F; Fischer, A; Magnussen, H; Coleman, R A

1995-11-01

The tachykinin-receptors mediating contraction of human bronchus have been characterized using both tachykinin-receptor selective agonists and blocking drugs under conditions where tachykinin metabolism by endogenous peptidases has been controlled, and true equilibrium conditions have been established. The findings that neurokinin A (EC50 = 2 nM) is the most potent agonist, and the NK2-receptor selective agonist, GR64349, is only 3-fold weaker, whereas agonists selective for NK1-receptors, substance P methyl ester, or NK3-receptors, senktide, are inactive, suggest that this effect is mediated exclusively by NK2-receptors. This is supported by observations that GR64349 is antagonised by the selective NK2-receptor blocking drugs, MEN10207 (pA2 = 6.7), R396 (pA2 = 6.1), (+/-)SR48968 (pA2 = 8.4) and GR159897 (pA2 = 8.6), but not by the NK1-receptor blocking drug, GR82334 (pA2 < 5). In approximately half of the preparations, the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM), caused a marked and well-maintained contraction (approximately 20% of neurokinin A maximum), which may indicate a role for endogenous tachykinins in the regulation of tone in this preparation. This is supported by the finding that neurokinin A-immunoreactive nerve fibres are located around intrinsic neurones of local ganglia and within the smooth muscle layer of this preparation.

Prefrontal cortical network activity: Opposite effects of psychedelic hallucinogens and D1/D5 dopamine receptor activation

PubMed Central

Lambe, Evelyn K.; Aghajanian, George K.

2007-01-01

The fine-tuning of network activity provides a modulating influence on how information is processed and interpreted in the brain. Here, we use brain slices of rat prefrontal cortex to study how recurrent network activity is affected by neuromodulators known to alter normal cortical function. We previously determined that glutamate spillover and stimulation of extrasynaptic NMDA receptors are required to support hallucinogen-induced cortical network activity. Since microdialysis studies suggest that psychedelic hallucinogens and dopamine D1/D5 receptor agonists have opposite effects on extracellular glutamate in prefrontal cortex, we hypothesized that these two families of psychoactive drugs would have opposite effects on cortical network activity. We found that network activity can be enhanced by DOI (a psychedelic hallucinogen that is a partial agonist of serotonin 5-HT2A/2C receptors) and suppressed by the selective D1/D5 agonist SKF 38393. This suppression could be mimicked by direct activation of adenylyl cyclase with forskolin or by addition of a cAMP analog. These findings are consistent with previous work showing that activation of adenylyl cyclase can upregulate neuronal glutamate transporters, thereby decreasing synaptic spillover of glutamate. Consistent with this hypothesis, a low concentration of the glutamate transporter inhibitor TBOA restored electrically-evoked recurrent activity in the presence of a selective D1/D5 agonist, whereas recurrent activity in the presence of a low level of the GABAA antagonist bicuculline was not resistant to suppression by the D1/D5 agonist. The tempering of network UP states by D1/D5 receptor activation may have implications for the proposed use of D1/D5 agonists in the treatment of schizophrenia. PMID:17293055

An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice.

PubMed

Montgomery, Megan D; Chan, Trevor; Swigart, Philip M; Myagmar, Bat-Erdene; Dash, Rajesh; Simpson, Paul C

2017-01-01

Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35-40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies.

The two-state dimer receptor model: a general model for receptor dimers.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2006-06-01

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

Pharmacological Studies of NOP Receptor Agonists as Novel Analgesics

DTIC Science & Technology

2009-05-01

found in hot-chili peppers that evokes pain sensation by activating at the TRPV1 . TRPV1 and the up-regulation of its expression have been strongly...via nociceptin/orphanin FQ receptors. Br J Pharmacol 137:1355-1361. Knotkova H, Pappagallo M, Szallasi A (2008) Capsaicin ( TRPV1 Agonist) therapy...Szallasi A, Cortright DN, Blum CA, Eid SR (2007). The vanilloid receptor TRPV1 : 10 years from channel cloning to antagonist proof of concept. Nat Rev Drug

Role of nitric oxide and septide-insensitive NK1 receptors in bronchoconstriction induced by aerosolised neurokinin A in guinea-pigs

PubMed Central

Ricciardolo, Fabio L M; Trevisani, Marcello; Geppetti, Pierangelo; Nadel, Jay A; Amadesi, Silvia; Bertrand, Claude

2000-01-01

The tachykinin, neurokinin A (NKA), contracts guinea-pig airways both in vitro and in vivo, preferentially activating smooth muscle NK2 receptors, although smooth muscle NK1 receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK1 receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK1 receptor agonists have been reported to activate both smooth muscle and epithelial NK1 receptors, however septide appears only to activate smooth muscle NK1 receptors. The aim of the present study was to investigate whether NKA-induced bronchoconstriction in guinea-pigs in vivo may be limited by NO release via NK1 receptor activation, and whether selective NK1 receptor agonists may activate this mechanism differently. Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK2 receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK1 receptor antagonist, CP-99,994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L-NAME, but not by the inactive enantiomer D-NAME. Potentiation by L-NAME of NKA-induced increase in RL was reversed by L-Arginine, but not by D-Arginine. Pretreatment with L-NAME did not affect the increase in RL induced by the selective NK2 receptor agonist, [β-Ala8]NKA(4-10), and by the selective NK1 receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK1 selective agonist, [Sar9,Met(O2)11]SP. Dose-response curves showed that septide was a more potent bronchoconstrictor than [Sar9,Met(O2)11]SP to cause bronchoconstriction. Pretreatment with the NK1 receptor antagonist, CP-96,994, abolished the ability of L-NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L-NAME, after pretreatment with the NK3 receptor antagonist, SR 142801. The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK1 receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea-pigs. PMID:10696090

Role of nitric oxide and septide-insensitive NK(1) receptors in bronchoconstriction induced by aerosolised neurokinin A in guinea-pigs.

PubMed

Ricciardolo, F L; Trevisani, M; Geppetti, P; Nadel, J A; Amadesi, S; Bertrand, C

2000-03-01

The tachykinin, neurokinin A (NKA), contracts guinea-pig airways both in vitro and in vivo, preferentially activating smooth muscle NK(2) receptors, although smooth muscle NK(1) receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK(1) receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK(1) receptor agonists have been reported to activate both smooth muscle and epithelial NK(1) receptors, however septide appears only to activate smooth muscle NK(1) receptors. The aim of the present study was to investigate whether NKA-induced bronchoconstriction in guinea-pigs in vivo may be limited by NO release via NK(1) receptor activation, and whether selective NK(1) receptor agonists may activate this mechanism differently. Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK(2) receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK(1) receptor antagonist, CP-99, 994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L-NAME, but not by the inactive enantiomer D-NAME. Potentiation by L-NAME of NKA-induced increase in RL was reversed by L-Arginine, but not by D-Arginine. Pretreatment with L-NAME did not affect the increase in RL induced by the selective NK(2) receptor agonist, [beta-Ala(8)]NKA(4-10), and by the selective NK(1) receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK(1) selective agonist, [Sar(9),Met(O(2))(11)]SP. Dose-response curves showed that septide was a more potent bronchoconstrictor than [Sar(9),Met(O(2))(11)]SP to cause bronchoconstriction. Pretreatment with the NK(1) receptor antagonist, CP-96,994, abolished the ability of L-NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L-NAME, after pretreatment with the NK(3) receptor antagonist, SR 142801. The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK(1) receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea-pigs.

The reducing agent dithiothreitol (DTT) does not abolish the inhibitory nicotinic response recorded from rat dorsolateral septal neurons

NASA Technical Reports Server (NTRS)

Sorenson, E. M.; Gallagher, J. P.

1993-01-01

Previous intracellular recordings have demonstrated that dorsolateral septal nucleus (DLSN) neurons express a novel nicotinic receptor which produces a direct membrane hyperpolarization when activated by nicotinic agonists. Activation of the classical excitatory nicotinic receptors has been shown to require a disulfide bond involving the cysteines at positions 192 and 193 of the alpha subunits of the receptor. Reduction of this cystine bond with dithiothreitol (DTT) abolishes agonist activation of excitatory nicotinic receptors. We have now examined whether DTT treatment of the inhibitory nicotinic receptor on DLSN neurons also abolishes the inhibitory nicotinic response. We find that the inhibitory response persists after treatment of the neurons with 1 mM DTT, even if the reduction is followed by alkylation of the receptor with bromoacetylcholine to prevent possible reformation of disulfide bonds. This result suggests that the agonist binding site on the inhibitory nicotinic receptor does not require an intact disulfide bond, similar to the bond on the alpha subunit of the excitatory nicotinic receptor, for agonist activation of the receptor. Some of these results have been previously reported in abstract form.

Effects of 5-HT1A Receptor Stimulation on D1 Receptor Agonist-Induced Striatonigral Activity and Dyskinesia in Hemiparkinsonian Rats

PubMed Central

2013-01-01

Accumulating evidence supports the value of 5-HT1A receptor (5-HT1AR) agonists for dyskinesias that arise with long-term L-DOPA therapy in Parkinson’s disease (PD). Yet, how 5-HT1AR stimulation directly influences the dyskinetogenic D1 receptor (D1R)-expressing striatonigral pathway remains largely unknown. To directly examine this, one cohort of hemiparkinsonian rats received systemic injections of Vehicle + Vehicle, Vehicle + the D1R agonist SKF81297 (0.8 mg/kg), or the 5-HT1AR agonist ±8-OH-DPAT (1.0 mg/kg) + SKF81297. Rats were examined for changes in abnormal involuntary movements (AIMs), rotations, striatal preprodynorphin (PPD), and glutamic acid decarboxylase (GAD; 65 and 67) mRNA via RT-PCR. In the second experiment, hemiparkinsonian rats received intrastriatal pretreatments of Vehicle (aCSF), ±8-OH-DPAT (7.5 mM), or ±8-OH-DPAT + the 5-HT1AR antagonist WAY100635 (4.6 mM), followed by systemic Vehicle or SKF81297 after which AIMs, rotations, and extracellular striatal glutamate and nigral GABA efflux were measured by in vivo microdialysis. Results revealed D1R agonist-induced AIMs were reduced by systemic and intrastriatal 5-HT1AR stimulation while rotations were enhanced. Although ±8-OH-DPAT did not modify D1R agonist-induced increases in striatal PPD mRNA, the D1R/5-HT1AR agonist combination enhanced GAD65 and GAD67 mRNA. When applied locally, ±8-OH-DPAT alone diminished striatal glutamate levels while the agonist combination increased nigral GABA efflux. Thus, presynaptic 5-HT1AR stimulation may attenuate striatal glutamate levels, resulting in diminished D1R-mediated dyskinetic behaviors, but maintain or enhance striatal postsynaptic factors ultimately increasing nigral GABA levels and rotational activity. The current findings offer a novel mechanistic explanation for previous results concerning 5-HT1AR agonists for the treatment of dyskinesia. PMID:23496922

Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

PubMed Central

Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

2014-01-01

The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor. PMID:24637012

Prospects of Using of κ-Opioid Receptor Agonists U-50,488 and ICI 199,441 for Improving Heart Resistance to Ischemia/Reperfusion.

PubMed

Tsibulnikov, S Yu; Maslov, L N; Mukhomedzyanov, A V; Krylatov, A V; Tsibulnikova, M R; Lishmanov, Yu B

2015-10-01

We studied the ability of the agonist of κ1-opioid receptors U-50,488 in doses of 0.1 and 1 mg/kg to simulate ischemic pre- and postconditioning of the heart and κ-opioid receptors ICI 199,441 in a dose of 0.1 mg/kg to simulate the antiarrhythmic effect of heart preconditioning. The duration of ischemia was 10 or 45 min and the duration of reperfusion was 10 min or 2 h. Administration of 1 mg/kg U-50,488 both before ischemia and 5 min before reperfusion produced a pronounced antiarrhythmic effect. U-50,488 injected 5 min before reperfusion 2-fold reduced the ratio of infarction to risk area. Administration of ICI 199,441 in a dose of 0.1 mg/kg 15 min before ischemia produced a potent antiarrhythmic effect. Antiarrhythmic effect of κ-opioid receptor agonists depended on activation of κ-opioid receptors.

Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington's Disease.

PubMed

Bagher, Amina M; Laprairie, Robert B; Kelly, Melanie E M; Denovan-Wright, Eileen M

2018-01-01

G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB 1 ) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB 1 levels and/or signaling may be beneficial for HD patients while decreasing CB 1 levels and/or signaling may be detrimental. Recent studies have found that Gα i/o -biased CB 1 agonists activate extracellular signal-regulated kinase (ERK), increase CB 1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB 1 agonists that are β-arrestin1-biased were found to reduce CB 1 protein levels and cell viability. Measuring agonist bias of known and novel CB 1 agonists will provide important data that predict CB 1 -specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes.

PubMed

Dallanoce, Clelia; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Roth, Bryan L; Ernsberger, Paul; De Micheli, Carlo

2007-12-15

A set of novel heterocyclic ligands (6-27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1-5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M(1), M(2), and M(3) tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC(50): 7.40 (M(1)), 8.18 (M(2)), and 8.14 (M(3))], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M(1) subtype [pK(B): 6.88 (M(1)), 5.95 (M(2)), 5.53 (M(3))]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M(1) antagonist/M(2) partial agonist/M(3) full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M(1-3) receptors, with an appreciable selectivity for the cardiac M(2) receptors.

Dopamine receptors play distinct roles in sexual behavior expression of rats with a different sexual motivational tone.

PubMed

Guadarrama-Bazante, Irma L; Canseco-Alba, Ana; Rodríguez-Manzo, Gabriela

2014-10-01

Dopamine (DA) plays a central role in the expression of male sexual behavior. The effects of DA-enhancing drugs on copulation seem to vary depending on the dose of the agonist used, the type of DA receptor activated, and the sexual condition of the animals. The aim of the present study was to carry out a systematic analysis of the effects of dopaminergic agonists on the expression of male sexual behavior by sexually competent rats in different sexual motivational states, that is when sexually active (sexually experienced) and when temporarily inhibited (sexually exhausted). To this end, the same doses of the nonselective DA receptor agonist apomorphine, the selective D2-like DA receptor agonist quinpirole, and the selective D1-like DA receptor agonist SKF38393 were injected intraperitoneally to sexually experienced or sexually exhausted male rats and their sexual behavior was recorded. Low apomorphine doses induced expression of sexual behavior in sexually satiated rats, but only reduced the intromission latency of sexually experienced rats. SKF38393 facilitated the expression of sexual behavior by sexually exhausted rats, but not that of sexually experienced males and quinpirole did not exert an effect in both types of animal. In line with these results, the apomorphine-induced reversal of sexual exhaustion was blocked by the D1-like receptor antagonist SCH23390. The data suggest that DA receptors play distinct roles in the expression of sexual behavior by male rats depending on their motivational state and that activation of D1-like receptors promotes the expression of sexual behavior in satiated rats.

An Allosteric Coagonist Model for Propofol Effects on α1β2γ2L γ-Aminobutyric Acid Type A Receptors

PubMed Central

Ruesch, Dirk; Neumann, Elena; Wulf, Hinnerk; Forman, Stuart A.

2011-01-01

Background Propofol produces its major actions via γ-aminobutyric acid type A (GABAA) receptors. At low concentrations, propofol enhances agonist-stimulated GABAA receptor activity, and high propofol concentrations directly activate receptors. Etomidate produces similar effects, and there is convincing evidence that a single class of etomidate sites mediate both agonist modulation and direct GABAA receptor activation. It is unknown if the propofol binding site(s) on GABAA receptors that modulate agonist-induced activity also mediate direct activation. Methods GABAA α1β2γ2L receptors were heterologously expressed in Xenopus oocytes and activity was quantified using voltage clamp electrophysiology. We tested whether propofol and etomidate display the same linkage between agonist modulation and direct activation of GABAA receptors by identifying equi-efficacious drug solutions for direct activation. We then determined whether these drug solutions produce equal modulation of GABA-induced receptor activity. We also measured propofol-dependent direct activation and modulation of low GABA responses. Allosteric coagonist models similar to that established for etomidate, but with variable numbers of propofol sites, were fitted to combined data. Results Solutions of 19 μM propofol and 10 μM etomidate were found to equally activate GABAA receptors. These two drug solutions also produced indistinguishable modulation of GABA-induced receptor activity. Combined electrophysiological data behaved in a manner consistent with allosteric co-agonist models with more than one propofol site. The best fit was observed when the model assumed three equivalent propofol sites. Conclusions Our results support the hypothesis that propofol, like etomidate, acts at GABAA receptor sites mediating both GABA modulation and direct activation. PMID:22104494

Molecular modelling studies on the ORL1-receptor and ORL1-agonists

NASA Astrophysics Data System (ADS)

Bröer, Britta M.; Gurrath, Marion; Höltje, Hans-Dieter

2003-11-01

The ORL1 ( opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.

Stereochemical studies of the monocyclic agouti-related protein (103-122) Arg-Phe-Phe residues: conversion of a melanocortin-4 receptor antagonist into an agonist and results in the discovery of a potent and selective melanocortin-1 agonist.

PubMed

Joseph, Christine G; Wang, Xiang S; Scott, Joseph W; Bauzo, Rayna M; Xiang, Zhimin; Richards, Nigel G; Haskell-Luevano, Carrie

2004-12-30

The agouti-related protein (AGRP) is an endogenous antagonist of the centrally expressed melanocortin receptors. The melanocortin-4 receptor (MC4R) is involved in energy homeostasis, food intake, sexual function, and obesity. The endogenous hAGRP protein is 132 amino acids in length, possesses five disulfide bridges at the C-terminus of the molecule, and is expressed in the hypothalamus of the brain. We have previously reported that a monocyclic hAGRP(103-122) peptide is an antagonist at the melanocortin receptors expressed in the brain. Stereochemical inversion from the endogenous l- to d-isomers of single or multiple amino acid modifications in this monocyclic truncated hAGRP sequence resulted in molecules that are converted from melanocortin receptor antagonists into melanocortin receptor agonists. The Asp-Pro-Ala-Ala-Thr-Ala-Tyr-cyclo[Cys-Arg-DPhe-DPhe-Asn-Ala-Phe-Cys]-Tyr-Ala-Arg-Lys-Leu peptide resulted in a 60 nM melanocortin-1 receptor agonist that is 100-fold selective versus the mMC4R, 1000-fold selective versus the mMC3R, and ca. 180-fold selective versus the mMC5R. In attempts to identify putative ligand-receptor interactions that may be participating in the agonist induced stimulation of the MC4R, selected ligands were docked into a homology molecular model of the mMC4R. These modeling studies have putatively identified hAGRP ligand DArg111-mMC4RAsn115 (TM3) and the hAGRP DPhe113-mMC4RPhe176 (TM4) interactions as important for agonist activity.

Electrophysiological evidence showing muscarinic agonist-antagonist activities of N-desmethylclozapine using hippocampal excitatory and inhibitory neurons.

PubMed

Sugawara, Yuto; Kikuchi, Yui; Yoneda, Mitsugu; Ohno-Shosaku, Takako

2016-07-01

The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K(+)current at -40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity. Copyright © 2016 Elsevier B.V. All rights reserved.

δ-Opioid Mechanisms for ADL5747 and ADL5859 Effects in Mice: Analgesia, Locomotion, and Receptor Internalization

PubMed Central

Nozaki, Chihiro; Le Bourdonnec, Bertrand; Reiss, David; Windh, Rolf T.; Little, Patrick J.; Dolle, Roland E.; Gavériaux-Ruff, Claire

2012-01-01

N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4′-piperidine]-4-yl) benzamide (ADL5859) and N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4′-piperidine]-4-yl)benzamide (ADL5747) are novel δ-opioid agonists that show good oral bioavailability and analgesic and antidepressive effects in the rat and represent potential drugs for chronic pain treatment. Here, we used genetic approaches to investigate molecular mechanisms underlying their analgesic effects in the mouse. We tested analgesic effects of ADL5859 and ADL5747 in mice by using mechanical sensitivity measures in both complete Freund's adjuvant and sciatic nerve ligation pain models. We examined their analgesic effects in δ-opioid receptor constitutive knockout (KO) mice and mice with a conditional deletion of δ-receptor in peripheral voltage-gated sodium channel (Nav)1.8-expressing neurons (cKO mice). Both ADL5859 and ADL5747, and the prototypical δ agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethyl-benzamide (SNC80) as a control, significantly reduced inflammatory and neuropathic pain. The antiallodynic effects of all three δ-opioid agonists were abolished in constitutive δ-receptor KO mice and strongly diminished in δ-receptor cKO mice. We also measured two other well described effects of δ agonists, increase in locomotor activity and agonist-induced receptor internalization by using knock-in mice expressing enhanced green fluorescence protein-tagged δ receptors. In contrast to SNC80, ADL5859 and ADL5747 did not induce either hyperlocomotion or receptor internalization in vivo. In conclusion, both ADL5859 and ADL5747 showed efficient pain-reducing properties in the two models of chronic pain. Their effects were mediated by δ-opioid receptors, with a main contribution of receptors expressed on peripheral Nav1.8-positive neurons. The lack of in vivo receptor internalization and locomotor activation, typically induced by SNC80, suggests agonist-biased activity at the receptor for the two drugs. PMID:22700431

The effects of dopamine receptor 1 and 2 agonists and antagonists on sexual and aggressive behaviors in male green anoles.

PubMed

Smith, Alexandra N; Kabelik, David

2017-01-01

The propensity to exhibit social behaviors during interactions with same-sex and opposite-sex conspecifics is modulated by various neurotransmitters, including dopamine. Dopamine is a conserved neurotransmitter among vertebrates and dopaminergic receptors are also highly conserved among taxa. Activation of D1 and D2 dopamine receptor subtypes has been shown to modulate social behaviors, especially in mammalian and avian studies. However, the specific behavioral functions of these receptors vary across taxa. In reptiles there have been few studies examining the relationship between dopaminergic receptors and social behaviors. We therefore examined the effects of D1 and D2 agonists and antagonists on sexual and aggressive behaviors in the male green anole lizard (Anolis carolinensis). Treatment with high doses of both D1 and D2 agonists was found to impair both sexual and aggressive behaviors. However, the D1 agonist treatment was also found to impair motor function, suggesting that those effects were likely nonspecific. Lower doses of both agonists and antagonists failed to affect social behaviors. These findings provide some evidence for D2 receptor regulation of social behaviors, but in contrast with previous research, these effects are all inhibitory and no effects were found for manipulations of D1 receptors. A potential reason for the lack of more widespread effects on social behaviors using moderate or low drug doses is that systemic injection of drugs resulted in effects throughout the whole brain, thus affecting counteracting circuits which negated one another, making measurable changes in behavioral output difficult to detect. Future studies should administer drugs directly into brain regions known to regulate sexual and aggressive behaviors.

Identification of functional bitter taste receptors and their antagonist in chickens.

PubMed

Dey, Bapon; Kawabata, Fuminori; Kawabata, Yuko; Yoshida, Yuta; Nishimura, Shotaro; Tabata, Shoji

2017-01-22

Elucidation of the taste sense of chickens is important not only for the development of chicken feedstuffs for the chicken industry but also to help clarify the evolution of the taste sense among animals. There are three putative chicken bitter taste receptors, chicken T2R1 (cT2R1), cT2R2 and cT2R7, which were identified using genome information and cell-based assays. Previously, we have shown that cT2R1 is a functional bitter taste receptor through both cell-based assays and behavioral tests. In this study, therefore, we focused on the sensitivities of the other two bitter receptors, cT2R2 and cT2R7, by using their agonists in behavioral tests. We tested three agonists of cT2R2 and three agonists of cT2R7. In a 10-min drinking study, the intakes of cT2R2 agonist solutions were not different from that of water. On the other hand, the intakes of cT2R7 agonist solutions were significantly lower compared to water. In addition, we constructed cT2R1-and cT2R7-expressing cells in order to search for an antagonist for these functional bitter taste receptors. By using Ca 2+ imaging methods, we found that 6-methoxyflavanone (6-meth) can inhibit the activities of both cT2R1 and cT2R7. Moreover, 6-meth also inhibited the reduction of the intake of bitter solutions containing cT2R1 or cT2R7 agonists in behavioral tests. Taken together, these results suggested that cT2R7 is a functional bitter taste receptor like cT2R1, but that cT2R2 is not, and that 6-meth is an antagonist for these two functional chicken bitter taste receptors. This is the first identification of an antagonist of chicken bitter receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

[18F]F15599, a novel 5-HT1A receptor agonist, as a radioligand for PET neuroimaging.

PubMed

Lemoine, Laëtitia; Verdurand, Mathieu; Vacher, Bernard; Blanc, Elodie; Le Bars, Didier; Newman-Tancredi, Adrian; Zimmer, Luc

2010-03-01

The serotonin-1A (5-HT(1A)) receptor is implicated in the pathophysiology of major neuropsychiatric disorders. Thus, the functional imaging of 5-HT(1A) receptors by positron emission tomography (PET) may contribute to the understanding of its role in those pathologies and their therapeutics. These receptors exist in high- and low-affinity states and it is proposed that agonists bind preferentially to the high-affinity state of the receptor and therefore could provide a measure of the functional 5-HT(1A) receptors. Since all clinical PET 5-HT(1A) radiopharmaceuticals are antagonists, it is of great interest to develop a( 18)F labelled agonist. F15599 (3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-methyl-pyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone) is a novel ligand with high affinity and selectivity for 5-HT(1A) receptors and is currently tested as an antidepressant. In pharmacological tests in rat, it exhibits preferential agonist activity at post-synaptic 5-HT(1A) receptors in cortical brain regions. Here, its nitro-precursor was synthesised and radiolabelled via a fluoronucleophilic substitution. Radiopharmacological evaluations included in vitro and ex vivo autoradiography in rat brain and PET scans on rats and cats. Results were compared with simultaneous studies using [(18)F]MPPF, a validated 5-HT(1A) antagonist radiopharmaceutical. The chemical and radiochemical purities of [(18)F]F15599 were >98%. In vitro [(18)F]F15599 binding was consistent with the known 5-HT(1A) receptors distribution (hippocampus, dorsal raphe nucleus, and notably cortical areas) and addition of Gpp(NH)p inhibited [(18)F]F15599 binding, consistent with a specific binding to G protein-coupled receptors. In vitro binding of [(18)F]F15599 was blocked by WAY100635 and 8-OH-DPAT, respectively, prototypical 5-HT(1A) antagonist and agonist. The ex vivo and in vivo studies demonstrated that the radiotracer readily entered the rat and the cat brain and generated few brain radioactive metabolites. Remarkably, in microPET studies, [(18)F]F15599 notably displayed a pattern of brain labelling that did not correlate with in vitro observations. Thus, in cat, the highest binding was observed in dorsal raphe and cingulate cortex with little binding in other cortical regions and none in hippocampus. In vivo binding was abolished by WAY100635, indicating specific labelling of 5-HT(1A) receptors. [(18)F]F15599 is a radiofluorinated agonist presenting interesting characteristics for probing in vitro and in vivo the high-affinity states of the 5-HT(1A) receptors. Its differential labelling of 5-HT(1A) receptors in vitro and in vivo may result from its reported preferential interaction with receptors coupled to specific G-protein subtypes.

Site and mechanism of the colokinetic action of the ghrelin receptor agonist, HM01.

PubMed

Naitou, K; Mamerto, T P; Pustovit, R V; Callaghan, B; Rivera, L R; Chan, A J; Ringuet, M T; Pietra, C; Furness, J B

2015-12-01

It has been recently demonstrated that the ghrelin receptor agonist, HM01, caused defecation in rats that were treated to provide a model for the constipation of Parkinson's disease. HM01 significantly increased fecal output and increased Fos activity in neurons of the hypothalamus and hindbrain, but not in the spinal defecation center. Other ghrelin agonists act on the defecation center. Receptor pharmacology was examined in ghrelin receptor (GHSR1a) transfected cells. Anesthetized rats were used to investigate sites and mechanisms of action. HM01 activated rat GHSR1a at nanomolar concentrations and was antagonized by the GHSR1a antagonist, YIL781. HM01, intravenous, was potent to activate propulsive colorectal contractions. This was prevented by pelvic nerve section and by intravenous YIL781, but not by spinal cord section rostral to the defecation centers. Direct intrathecal application of HM01 to the defecation center at spinal level L6-S1 initiated propulsive contractions of the colorectum. HM01 stimulates GHSR1a receptors on neurons in the lumbosacral defecation centers to cause propulsive contractions and emptying of the colorectum. It has greater potency when given systemically, compared with other GHSR1a agonists. © 2015 John Wiley & Sons Ltd.

The modulatory role of spinally located histamine receptors in the regulation of the blood glucose level in d-glucose-fed mice.

PubMed

Sim, Yun-Beom; Park, Soo-Hyun; Kim, Sung-Su; Kim, Chea-Ha; Kim, Su-Jin; Lim, Su-Min; Jung, Jun-Sub; Ryu, Ohk-Hyun; Choi, Moon-Gi; Suh, Hong-Won

2014-02-01

The possible roles of spinal histamine receptors in the regulation of the blood glucose level were studied in ICR mice. Mice were intrathecally (i.t.) treated with histamine 1 (H1) receptor agonist (2-pyridylethylamine) or antagonist (cetirizine), histamine 2 (H2) receptor agonist (dimaprit) or antagonist (ranitidine), histamine 3 (H3) receptor agonist (α-methylhistamine) or antagonist (carcinine) and histamine 4 (H4) receptor agonist (VUF 8430) or antagonist (JNJ 7777120), and the blood glucose level was measured at 30, 60 and 120 min after i.t. administration. The i.t. injection with α-methylhistamine, but not carcinine slightly caused an elevation of the blood glucose level. In addition, histamine H1, H2, and H4 receptor agonists and antagonists did not affect the blood glucose level. In D-glucose-fed model, i.t. pretreatment with cetirizine enhanced the blood glucose level, whereas 2-pyridylethylamine did not affect. The i.t. pretreatment with dimaprit, but not ranitidine, enhanced the blood glucose level in D-glucose-fed model. In addition, α-methylhistamine, but not carcinine, slightly but significantly enhanced the blood glucose level D-glucose-fed model. Finally, i.t. pretreatment with JNJ 7777120, but not VUF 8430, slightly but significantly increased the blood glucose level. Although histamine receptors themselves located at the spinal cord do not exert any effect on the regulation of the blood glucose level, our results suggest that the activation of spinal histamine H2 receptors and the blockade of spinal histamine H1 or H3 receptors may play modulatory roles for up-regulation and down-regulation, respectively, of the blood glucose level in D-glucose fed model.

Dopamine D3 Receptors Mediate the Discriminative Stimulus Effects of Quinpirole in Free-Feeding Rats

PubMed Central

Baladi, Michelle G.; Newman, Amy H.

2010-01-01

The discriminative stimulus effects of dopamine (DA) D3/D2 receptor agonists are thought to be mediated by D2 receptors. To maintain responding, access to food is often restricted, which can alter neurochemical and behavioral effects of drugs acting on DA systems. This study established stimulus control with quinpirole in free-feeding rats and tested the ability of agonists to mimic and antagonists to attenuate the effects of quinpirole. The same antagonists were studied for their ability to attenuate quinpirole-induced yawning and hypothermia. DA receptor agonists apomorphine and lisuride, but not amphetamine and morphine, occasioned responding on the quinpirole lever. The discriminative stimulus effects of quinpirole were attenuated by the D3 receptor-selective antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl (PG01037) and the nonselective D3/D2 receptor antagonist raclopride, but not by the D2 receptor-selective antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl-1H-indole (L-741,626); the potencies of PG01037 and raclopride to antagonize this effect of quinpirole paralleled their potencies to antagonize the ascending limb of the quinpirole yawning dose-response curve (thought to be mediated by D3 receptors). L-741,626 selectively antagonized the descending limb of the quinpirole yawning dose-response curve, and both L-741,626 and raclopride, but not PG01037, antagonized the hypothermic effects of quinpirole (thought to be mediated by D2 receptors). Food restriction (10 g/day/7 days) significantly decreased quinpirole-induced yawning without affecting the quinpirole discrimination. Many discrimination studies on DA receptor agonists use food-restricted rats; together with those studies, the current experiment using free-feeding rats suggests that feeding conditions affecting the behavioral effects of direct-acting DA receptor agonists might also have an impact on the effects of indirect-acting agonists such as cocaine and amphetamine. PMID:19797621

Dopamine D3 receptors mediate the discriminative stimulus effects of quinpirole in free-feeding rats.

PubMed

Baladi, Michelle G; Newman, Amy H; France, Charles P

2010-01-01

The discriminative stimulus effects of dopamine (DA) D3/D2 receptor agonists are thought to be mediated by D2 receptors. To maintain responding, access to food is often restricted, which can alter neurochemical and behavioral effects of drugs acting on DA systems. This study established stimulus control with quinpirole in free-feeding rats and tested the ability of agonists to mimic and antagonists to attenuate the effects of quinpirole. The same antagonists were studied for their ability to attenuate quinpirole-induced yawning and hypothermia. DA receptor agonists apomorphine and lisuride, but not amphetamine and morphine, occasioned responding on the quinpirole lever. The discriminative stimulus effects of quinpirole were attenuated by the D3 receptor-selective antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl (PG01037) and the nonselective D3/D2 receptor antagonist raclopride, but not by the D2 receptor-selective antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl-1H-indole (L-741,626); the potencies of PG01037 and raclopride to antagonize this effect of quinpirole paralleled their potencies to antagonize the ascending limb of the quinpirole yawning dose-response curve (thought to be mediated by D3 receptors). L-741,626 selectively antagonized the descending limb of the quinpirole yawning dose-response curve, and both L-741,626 and raclopride, but not PG01037, antagonized the hypothermic effects of quinpirole (thought to be mediated by D2 receptors). Food restriction (10 g/day/7 days) significantly decreased quinpirole-induced yawning without affecting the quinpirole discrimination. Many discrimination studies on DA receptor agonists use food-restricted rats; together with those studies, the current experiment using free-feeding rats suggests that feeding conditions affecting the behavioral effects of direct-acting DA receptor agonists might also have an impact on the effects of indirect-acting agonists such as cocaine and amphetamine.

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

PubMed

Hashi, Hiroki; Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

2018-04-01

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of 3-aryl-4-isoxazolecarboxamides as TGR5 receptor agonists.

PubMed

Evans, Karen A; Budzik, Brian W; Ross, Sean A; Wisnoski, David D; Jin, Jian; Rivero, Ralph A; Vimal, Mythily; Szewczyk, George R; Jayawickreme, Channa; Moncol, David L; Rimele, Thomas J; Armour, Susan L; Weaver, Susan P; Griffin, Robert J; Tadepalli, Sarva M; Jeune, Michael R; Shearer, Todd W; Chen, Zibin B; Chen, Lihong; Anderson, Donald L; Becherer, J David; De Los Frailes, Maite; Colilla, Francisco Javier

2009-12-24

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

Discovery of novel brain permeable and G protein-biased beta-1 adrenergic receptor partial agonists for the treatment of neurocognitive disorders

PubMed Central

Yi, Bitna; Jahangir, Alam; Evans, Andrew K.; Briggs, Denise; Ravina, Kristine; Ernest, Jacqueline; Farimani, Amir B.; Sun, Wenchao; Rajadas, Jayakumar; Green, Michael; Feinberg, Evan N.; Pande, Vijay S.

2017-01-01

The beta-1 adrenergic receptor (ADRB1) is a promising therapeutic target intrinsically involved in the cognitive deficits and pathological features associated with Alzheimer’s disease (AD). Evidence indicates that ADRB1 plays an important role in regulating neuroinflammatory processes, and activation of ADRB1 may produce neuroprotective effects in neuroinflammatory diseases. Novel small molecule modulators of ADRB1, engineered to be highly brain permeable and functionally selective for the G protein with partial agonistic activity, could have tremendous value both as pharmacological tools and potential lead molecules for further preclinical development. The present study describes our ongoing efforts toward the discovery of functionally selective partial agonists of ADRB1 that have potential therapeutic value for AD and neuroinflammatory disorders, which has led to the identification of the molecule STD-101-D1. As a functionally selective agonist of ADRB1, STD-101-D1 produces partial agonistic activity on G protein signaling with an EC50 value in the low nanomolar range, but engages very little beta-arrestin recruitment compared to the unbiased agonist isoproterenol. STD-101-D1 also inhibits the tumor necrosis factor α (TNFα) response induced by lipopolysaccharide (LPS) both in vitro and in vivo, and shows high brain penetration. Other than the therapeutic role, this newly identified, functionally selective, partial agonist of ADRB1 is an invaluable research tool to study mechanisms of G protein-coupled receptor signal transduction. PMID:28746336

Discovery of novel brain permeable and G protein-biased beta-1 adrenergic receptor partial agonists for the treatment of neurocognitive disorders.

PubMed

Yi, Bitna; Jahangir, Alam; Evans, Andrew K; Briggs, Denise; Ravina, Kristine; Ernest, Jacqueline; Farimani, Amir B; Sun, Wenchao; Rajadas, Jayakumar; Green, Michael; Feinberg, Evan N; Pande, Vijay S; Shamloo, Mehrdad

2017-01-01

The beta-1 adrenergic receptor (ADRB1) is a promising therapeutic target intrinsically involved in the cognitive deficits and pathological features associated with Alzheimer's disease (AD). Evidence indicates that ADRB1 plays an important role in regulating neuroinflammatory processes, and activation of ADRB1 may produce neuroprotective effects in neuroinflammatory diseases. Novel small molecule modulators of ADRB1, engineered to be highly brain permeable and functionally selective for the G protein with partial agonistic activity, could have tremendous value both as pharmacological tools and potential lead molecules for further preclinical development. The present study describes our ongoing efforts toward the discovery of functionally selective partial agonists of ADRB1 that have potential therapeutic value for AD and neuroinflammatory disorders, which has led to the identification of the molecule STD-101-D1. As a functionally selective agonist of ADRB1, STD-101-D1 produces partial agonistic activity on G protein signaling with an EC50 value in the low nanomolar range, but engages very little beta-arrestin recruitment compared to the unbiased agonist isoproterenol. STD-101-D1 also inhibits the tumor necrosis factor α (TNFα) response induced by lipopolysaccharide (LPS) both in vitro and in vivo, and shows high brain penetration. Other than the therapeutic role, this newly identified, functionally selective, partial agonist of ADRB1 is an invaluable research tool to study mechanisms of G protein-coupled receptor signal transduction.

Agonist Met antibodies define the signalling threshold required for a full mitogenic and invasive program of Kaposi's Sarcoma cells.

PubMed

Bardelli, Claudio; Sala, Marilena; Cavallazzi, Umberto; Prat, Maria

2005-09-09

We previously showed that the Kaposi Sarcoma line KS-IMM express a functional Met tyrosine kinase receptor, which, upon HGF stimulation, activates motogenic, proliferative, and invasive responses. In this study, we investigated the signalling pathways activated by HGF, as well as by Met monoclonal antibodies (Mabs), acting as full or partial agonists. The full agonist Mab mimics HGF in all biological and biochemical aspects. It elicits the whole spectrum of responses, while the partial agonist Mab induces only wound healing. These differences correlated with a more prolonged and sustained tyrosine phosphorylation of the receptor and MAPK evoked by HGF and by the full agonist Mab, relative to the partial agonist Mab. Since Gab1, JNK and PI 3-kinase are activated with same intensity and kinetics by HGF and by the two agonist antibodies, it is concluded that level and duration of MAPK activation by Met receptor are crucial for the induction of a full HGF-dependent mitogenic and invasive program in KS cells.

Acute and Delayed Systemic Treatment with Cannabinoid Receptor 2 Agonists to Prevent or Treat/Reverse Osteoporosis in a Mouse Model of SCI

DTIC Science & Technology

2017-08-01

AWARD NUMBER: W81XWH-16-1-0349 TITLE: Acute and Delayed Systemic Treatment with Cannabinoid Receptor 2 Agonists to Prevent or Treat/Reverse...REPORT TYPE Annual 3. DATES COVERED 1 Aug 2016 - 31 Jul 2017 4. TITLE AND SUBTITLE Acute and Delayed Systemic Treatment with Cannabinoid Receptor 2...for the cannabinoid-2 receptor, when systemically delivered, can prevent the onset of osteoporosis in mice when delivered during the acute phase of

Investigation of the interaction between the atypical agonist c[YpwFG] and MOR.

PubMed

Gentilucci, Luca; Squassabia, Federico; De Marco, Rossella; Artali, Roberto; Cardillo, Giuliana; Tolomelli, Alessandra; Spampinato, Santi; Bedini, Andrea

2008-05-01

Endogenous and exogenous opiates are currently considered the drugs of choice for treating different kinds of pain. However, their prolonged use produces several adverse symptoms, and in addition, many forms of pain are resistant to any kind of therapy. Therefore, the discovery of compounds active towards mu-opioid receptors (MORs) by alternative pharmacological mechanisms could be of value for developing novel classes of analgesics. There is evidence that some unusual molecules can bind opioid receptors, albeit lacking some of the typical opioid pharmacophoric features. In particular, the recent discovery of a few compounds that showed agonist behavior even in the absence of the primary pharmacophore, namely a protonable amine, led to a rediscussion of the importance of ionic interactions in stabilizing the ligand-receptor complex and in activating signal transduction. Very recently, we synthesized a library of cyclic analogs of the endogenous, MOR-selective agonist endomorphin-1 (YPWF-NH(2)), containing a Gly5 bridge between Tyr1 and Phe4. The cyclopeptide c[YpwFG] showed good affinity and agonist behavior. This atypical MOR agonist does not have the protonable Tyr amine. In order to gain more information about plausible mechanisms of interaction between c[YpwFG] and the opioid receptor, we synthesized a selected set of derivatives containing different bridges between Tyr1 and Phe4, and tested their affinities towards mu-opioid receptors. We performed conformational analysis of the cyclopeptides by NMR spectroscopy and molecular dynamics, and investigated plausible, unprecedented modes of interaction with the MOR by molecular docking. The successive quantum mechanics/molecular mechanics investigation of the complexes obtained by the molecular docking procedure furnished a more detailed description of the binding mode and the electronic properties of the ligands. The comparison with the binding mode of the potent agonist JOM-6 seems to indicate that the cyclic endomorphin-1 analogs interact with the receptor by way of an alternative mechanism, still maintaining the ability to activate the receptor.

Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor.

PubMed

Troppmann, B; Balfanz, S; Baumann, A; Blenau, W

2010-04-01

5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT(1) receptor of an insect model for neurobiology, physiology and pharmacology. A cDNA encoding for the Periplaneta americana 5-HT(1) receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. The P. americana 5-HT(1) receptor (Pea5-HT(1)) shares pronounced sequence and functional similarity with mammalian 5-HT(1) receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT(1) was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT(1) receptor. The results presented here should facilitate further analyses of 5-HT(1) receptors in mediating central and peripheral effects of 5-HT in insects.

The Glycine Transport Inhibitor Sarcosine Is an Inhibitory Glycine Receptor Agonist

PubMed Central

Zhang, Hai Xia; Lyons-Warren, Ariel; Thio, Liu Lin

2009-01-01

Summary Sarcosine is an endogenous amino acid that is a competitive inhibitor of the type I glycine transporter (GlyT1), an N-methyl-D-aspartate receptor (NMDAR) co-agonist, and an important intermediate in one-carbon metabolism. Its therapeutic potential for schizophrenia further underscores its clinical importance. The structural similarity between sarcosine and glycine and sarcosine's ability to serve as an NMDAR co-agonist led us to examine whether sarcosine is also an agonist at the inhibitory glycine receptor (GlyR). We examined this possibility using whole-cell recordings from cultured embryonic mouse hippocampal neurons and found that sarcosine evoked a dose-dependent, strychnine sensitive, Cl- current that cross-inhibited glycine currents. Sarcosine evoked this current with Li+ in the extracellular solution to block GlyT1, in neurons treated with the essentially irreversible GlyT1 inhibitor N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS), and in neurons plated in the absence of glia. These results indicate that the sarcosine currents did not result from GlyT1 inhibition or heteroexchange. We conclude that sarcosine is a GlyR agonist. PMID:19619564

The glycine transport inhibitor sarcosine is an inhibitory glycine receptor agonist.

PubMed

Zhang, Hai Xia; Lyons-Warren, Ariel; Thio, Liu Lin

2009-01-01

Sarcosine is an endogenous amino acid that is a competitive inhibitor of the type I glycine transporter (GlyT1), an N-methyl-d-aspartate receptor (NMDAR) co-agonist, and an important intermediate in one-carbon metabolism. Its therapeutic potential for schizophrenia further underscores its clinical importance. The structural similarity between sarcosine and glycine and sarcosine's ability to serve as an NMDAR co-agonist led us to examine whether sarcosine is also an agonist at the inhibitory glycine receptor (GlyR). We examined this possibility using whole-cell recordings from cultured embryonic mouse hippocampal neurons and found that sarcosine evoked a dose-dependent, strychnine sensitive, Cl(-) current that cross-inhibited glycine currents. Sarcosine evoked this current with Li(+) in the extracellular solution to block GlyT1, in neurons treated with the essentially irreversible GlyT1 inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), and in neurons plated in the absence of glia. These results indicate that the sarcosine currents did not result from GlyT1 inhibition or heteroexchange. We conclude that sarcosine is a GlyR agonist.

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

PubMed

Bianchi, B R; Lynch, K J; Touma, E; Niforatos, W; Burgard, E C; Alexander, K M; Park, H S; Yu, H; Metzger, R; Kowaluk, E; Jarvis, M F; van Biesen, T

1999-07-02

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Vasopressin-induced constriction of the isolated rat occipital artery is segment-dependent

PubMed Central

Chelko, Stephen P.; Schmiedt, Chad W.; Lewis, Tristan H.; Lewis, Stephen J.; Robertson, Tom P.

2014-01-01

Background Circulating factors delivered to the nodose ganglion (NG) by the occipital artery (OA) have shown to affect vagal afferent activity, and thus the contractile state of the OA may influence blood flow to the NG. Methods OA were isolated and bisected into proximal and distal segments, relative to the external carotid artery. Results Bisection, highlighted stark differences between maximal contractile responses and OA sensitivity. Specifically, maximum responses to vasopressin and the V1 receptor agonist, were significantly higher in distal than proximal segments. Distal segments were significantly more sensitive to 5-HT and the 5-HT2 receptor agonist than proximal segments. AT2, V2 and 5-HT1B/1D receptor agonists did not elicit vascular responses. Additionally, AT1 receptor agonists elicited mild, yet not significantly different maximal responses between segments. Conclusion The results of this study are consistent with contractile properties of rat OA being mediated via AT1, V1 and 5-HT2 receptors, and are dependent upon the OA segment. Furthermore, vasopressin-induced constriction of the OA, regardless of a bolus dose or a first and second concentration response curve retained this unique segmental difference and therefore we hypothesize this may be a pathophysiological response in the regulation of blood flow through the OA. PMID:24192548

Potentiation of adenosine A1 receptor agonist CPA-induced antinociception by paeoniflorin in mice.

PubMed

Liu, Da-Zhi; Zhao, Fei-Li; Liu, Jing; Ji, Xin-Quan; Ye, Yang; Zhu, Xing-Zu

2006-08-01

The effect of paeoniflorin (PF), a major constituent isolated from Paeony radix, on N6-Cyclopentyladenosine (CPA), a selective adenosine A1 receptor (A1 receptor) agonist, induced antinociception was examined in mice. In the tail-pressure test, CPA (0.05, 0.1, 0.2 mg/kg, s.c.) could induce antinociception in a dose-dependent manner. PF (5, 10, 20 mg/kg, s.c.) alone failed to exhibit any antinociceptive effect in mice; however, pretreatment of PF (20 mg/kg, s.c.) could significantly enhance CPA-induced antinociception. Additionally, pretreatment of 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.25 mg/kg, s.c.), a selective A1 receptor antagonist, could antagonize the antinociceptive effect of combining CPA with PF. Furthermore, in the competitive binding experiments, PF did not displace the binding of [3H]-8-Cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) but displaced that of [3H]-2-Chloro-N6-cyclopentyladenosine ([3H]-CCPA, a selective A1 receptor agonist) to the membrane preparation of rat cerebral cortex. These results suggested that PF might selectively increase the binding and antinociceptive effect of CPA by binding with A1 receptor.

Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells.

PubMed

Reagan, L P; Ye, X; Maretzski, C H; Fluharty, S J

1993-01-01

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Enrichment assessment of multiple virtual screening strategies for Toll-like receptor 8 agonists based on a maximal unbiased benchmarking data set.

PubMed

Pei, Fen; Jin, Hongwei; Zhou, Xin; Xia, Jie; Sun, Lidan; Liu, Zhenming; Zhang, Liangren

2015-11-01

Toll-like receptor 8 agonists, which activate adaptive immune responses by inducing robust production of T-helper 1-polarizing cytokines, are promising candidates for vaccine adjuvants. As the binding site of toll-like receptor 8 is large and highly flexible, virtual screening by individual method has inevitable limitations; thus, a comprehensive comparison of different methods may provide insights into seeking effective strategy for the discovery of novel toll-like receptor 8 agonists. In this study, the performance of knowledge-based pharmacophore, shape-based 3D screening, and combined strategies was assessed against a maximum unbiased benchmarking data set containing 13 actives and 1302 decoys specialized for toll-like receptor 8 agonists. Prior structure-activity relationship knowledge was involved in knowledge-based pharmacophore generation, and a set of antagonists was innovatively used to verify the selectivity of the selected knowledge-based pharmacophore. The benchmarking data set was generated from our recently developed 'mubd-decoymaker' protocol. The enrichment assessment demonstrated a considerable performance through our selected three-layer virtual screening strategy: knowledge-based pharmacophore (Phar1) screening, shape-based 3D similarity search (Q4_combo), and then a Gold docking screening. This virtual screening strategy could be further employed to perform large-scale database screening and to discover novel toll-like receptor 8 agonists. © 2015 John Wiley & Sons A/S.

The effects of acute multiple intraperitoneal injections of the GABAB receptor agonist baclofen on food intake in rats.

PubMed

Patel, Sunit M; Ebenezer, Ivor S

2008-12-28

This study was undertaken to examine the effects of acute repeated administration of the GABA(B) receptor agonist baclofen on food intake in rats. In Experiment 1, the effects of repeated intraperitoneal (i.p.) injections of the GABA(B) receptor agonist baclofen (1 and 2 mg/kg) at 2 h intervals were investigated on food intake in non-deprived male Wistar rats. Both doses of baclofen significantly increased food intake after the 1st injection (P<0.05), but had no effects on intake following the 2nd and 3rd injections. By contrast, in Experiment 2, diazepam (1 and 2 mg/kg, i.p.) significantly increased food intake (at least, P<0.05) after each of 3 injection separated by 2 h in non-deprived rats. These data show that tolerance occurs to the hyperphagic effects of baclofen with acute multiple injections, and may have important implications for future studies investigating the effects of GABA(B) receptor agonists on food intake and energy homeostasis.

Group III mGlu receptor agonists potentiate the anticonvulsant effect of AMPA and NMDA receptor block.

PubMed

De Sarro, Giovambattista; Chimirri, Alba; Meldrum, Brian S

2002-09-06

We report the anticonvulsant action in DBA/2 mice of two mGlu Group III receptor agonists: (R,S)-4-phosphonophenylglycine, (R,S)-PPG, a compound with moderate mGlu8 selectivity, and of (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid, ACPT-1, a selective agonist for mGlu4alpha receptors. Both compounds, given intracerebroventricularly at doses which did not show marked anticonvulsant activity, produced a consistent shift to the left of the dose-response curves (i.e. enhanced the anticonvulsant properties) of 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one hydrochloride, CFM-2, a noncompetitive AMPA receptor antagonist, and 3-((+/-)-2-carboxypiperazin-4-yl)-1-phosphonic acid, CPPene, a competitive NMDA receptor antagonist, in DBA/2 mice. In addition, (R,S)-PPG and ACPT-1 administered intracerebroventricularly prolonged the time course of the anticonvulsant properties of CFM-2 (33 micromol/kg, i.p.) and CPPene (3.3 micromol/kg, i.p.) administered intraperitoneally. We conclude that modest reduction of synaptic glutamate release by activation of Group III metabotropic receptors potentiates the anticonvulsant effect of AMPA and NMDA receptor blockade. Copyright 2002 Elsevier Science B.V.

Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway

PubMed Central

Wang, Anping; Li, Ting; An, Ping; Yan, Wenhua; Zheng, Hua; Wang, Baoan; Mu, Yiming

2017-01-01

Glucagon-like peptide-1 (GLP-1) receptor plays an essential role in regulating glucose metabolism. GLP-1 receptor agonists have been widely used for treating diabetes and other insulin resistance-related diseases. However, mechanisms underlying the anti-diabetic effects of GLP-1 receptor agonists remain largely unknown. In this study, we investigated the effects of GLP-1 agonist exendin-4 on the expression of adiponectin, an insulin sensitizing hormone. We found that exendin-4 increased the expression and secretion of adiponectin both in vitro and in vivo. Our data showed that exendin-4 upregulated adiponectin expression at both mRNA and protein levels in adipocytes and adipose tissues. The effects of exendin-4 on adiponectin expression were dependent on the GLP-1 receptor. We further demonstrated important roles of Sirt1 and transcriptional factor Foxo-1 in mediating the function of exendin-4 in regulating adiponectin expression. Suppression of Sirt1 or Foxo-1 expression significantly impaired exendin-4-induced adiponectin expression. Consistently, exendin-4 up-regulated Sirt1 and Foxo-1 expression in vivo. Our work is the first study demonstrating the role of Sirt1/Foxo-1 in regulating the regulatory function of a GLP-1 receptor agonist in adiponectin expression both in vitro and in vivo. The results provide important information for the mechanism underlying the function of GLP-1R on improving insulin resistance and related diseases. PMID:28122026

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

PubMed Central

Bower, Kiowa S.; Price, Kerry L.; Sturdee, Laura E.C.; Dayrell, Mariza; Dougherty, Dennis A.; Lummis, Sarah C.R.

2008-01-01

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax / Imax5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50 = 16 and 27 μM, and Ki for displacement of [3H]granisetron binding = 0.8 and 1.8 μM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax = 0.15), low affinity (EC50 = 113 μM; Ki = 4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50 = 8.1 μM; Ki = 2.7 μM) but is a very weak partial agonist (Rmax = 0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening. PMID:18082160

Hyperactivity induced by stimulation of separate dopamine D-1 and D-2 receptors in rats with bilateral 6-OHDA lesions.

PubMed

Arnt, J

1985-08-26

The effects of DA agonists and antagonists with different dopamine (DA) D-1 and D-2 receptor selectivity have been studied in rats with bilateral 6-OHDA lesions. The D-1 agonist SK & F 38393, the D-2 agonist pergolide and the mixed agonist apomorphine all induced marked hyperactivity in lesioned rats in doses which were without stimulant effect in sham-operated animals. The hyperactivity induced by SK & F 38393 was blocked by the DA D-1 antagonist SCH 23390, but unaffected by the D-2 antagonists spiroperidol or clebopride. Pergolide-induced hyperactivity showed the reverse selectivity. The mixed D-1/D-2 antagonists, cis(Z)-flupentixol and cis(Z)-clopenthixol, however blocked the effect of both agonists. Apomorphine-induced hyperactivity was neither blocked by selective D-1 nor D-2 antagonists, but was dose-dependently inhibited by cis(Z)-flupentixol and cis(Z)-clopenthixol. Potent blockade was also obtained by combined treatment with SCH 23390 and spiroperidol, indicating the need of blocking both D-1 and D-2 receptors simultaneously. The results indicate that D-1 and D-2 receptor function can be independently manipulated in denervated rats and they confirm similar results obtained in rats with unilateral 6-OHDA lesions using circling behaviour.

GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

PubMed Central

Ray, N. J.; Jones, A. J.; Keen, P.

1991-01-01

1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-)-baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane-sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP-LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma. PMID:1713105

Cholinergic agonists increase intracellular calcium concentration in frog vestibular hair cells.

PubMed

Ohtani, M; Devau, G; Lehouelleur, J; Sans, A

1994-11-01

Acetylcholine (ACh) is usually considered to be the neurotransmitter of the efferent vestibular system. The nature and the localization of cholinergic receptors have been investigated on frog isolated vestibular hair cells (VHCs), by measuring variations of intracellular calcium concentration ([Ca2+]i), using calcium sensitive dye fura-2. Focal iontophoretic ACh (1 M, 300 nA.40 ms) application induced a rapid increase in [Ca2+]i, reaching a peak in 20 s and representing about 5-fold the resting level (from 61 +/- 6 to 320 +/- 26 nM). Applications of muscarinic agonists as methacholine and carbachol induced weaker calcium responses (from 78 +/- 25 to 238 +/- 53 nM) than the one obtained with ACh applications. These muscarinic agonists were efficient only in precise zones. Desensitization of muscarinic receptors to successive stimulations was significant. Perfusion of nicotine or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic agonist, induced an increase in [Ca2+]i only in some cells (4/28 with DMPP). These results indicated the presence of cholinergic receptors on frog VHCs: muscarinic receptors were more responsive than nicotinic receptors. Presence of muscarinic and nicotinic receptors in the membrane of VHCs could indicate different modulations of VHCs activity mediated by [Ca2+]i and involving an efferent control which represents a central regulation of the vestibular afferent message.

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor.

PubMed

Ecke, Denise; Hanck, Theodor; Tulapurkar, Mohan E; Schäfer, Rainer; Kassack, Matthias; Stricker, Rolf; Reiser, Georg

2008-01-01

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

Agomelatine or ramelteon as treatment adjuncts in glioblastoma and other M1- or M2-expressing cancers

PubMed Central

2015-01-01

The impressive but sad list of over forty clinical studies using various cytotoxic chemotherapies published in the last few years has failed to increase median survival of glioblastoma beyond two years after diagnosis. In view of this apparent brick wall, adjunctive non-cytotoxic growth factor blocking drugs are being tried, as in the CUSP9* protocol. A related theme is searching for agonists at growth inhibiting receptors. One such dataset is that of melatonin agonism at M1 or M2 receptors found on glioblastoma cells, being a negative regulator of these cells’ growth. Melatonin itself is an endogenous hormone, but when used as an exogenously administered drug it has many disadvantages. Agomelatine, marketed as an antidepressant, and ramelteon, marketed as a treatment for insomnia, are currently-available melatonin receptor agonists. These melatonin receptor agonists have significant advantages over the natural ligand: longer half-life, better oral absorption, and higher affinity to melatonin receptors. They have an eminently benign side effect profile. As full agonists they should function to inhibit glioblastoma growth, as demonstrated for melatonin. A potentially helpful ancillary attribute of melatonergic agonists in glioblastoma treatment is an increase in interleukin-2 synthesis, expected, at least partially, to reverse some of the immunosuppression associated with glioblastoma. PMID:26034396

Agomelatine or ramelteon as treatment adjuncts in glioblastoma and other M1- or M2-expressing cancers.

PubMed

Kast, Richard E

2015-01-01

The impressive but sad list of over forty clinical studies using various cytotoxic chemotherapies published in the last few years has failed to increase median survival of glioblastoma beyond two years after diagnosis. In view of this apparent brick wall, adjunctive non-cytotoxic growth factor blocking drugs are being tried, as in the CUSP9* protocol. A related theme is searching for agonists at growth inhibiting receptors. One such dataset is that of melatonin agonism at M1 or M2 receptors found on glioblastoma cells, being a negative regulator of these cells' growth. Melatonin itself is an endogenous hormone, but when used as an exogenously administered drug it has many disadvantages. Agomelatine, marketed as an antidepressant, and ramelteon, marketed as a treatment for insomnia, are currently-available melatonin receptor agonists. These melatonin receptor agonists have significant advantages over the natural ligand: longer half-life, better oral absorption, and higher affinity to melatonin receptors. They have an eminently benign side effect profile. As full agonists they should function to inhibit glioblastoma growth, as demonstrated for melatonin. A potentially helpful ancillary attribute of melatonergic agonists in glioblastoma treatment is an increase in interleukin-2 synthesis, expected, at least partially, to reverse some of the immunosuppression associated with glioblastoma.

Convergent Pathways for Steroid Hormone-and Neurotransmitter-Induced Rat Sexual Behavior

NASA Astrophysics Data System (ADS)

Mani, S. K.; Allen, J. M. C.; Clark, J. H.; Blaustein, J. D.; O'Malley, B. W.

1994-08-01

Estrogen and progesterone modulate gene expression in rodents by activation of intracellular receptors in the hypothalamus, which regulate neuronal networks that control female sexual behavior. However, the neurotransmitter dopamine has been shown to activate certain steroid receptors in a ligand-independent manner. A dopamine receptor stimulant and a D_1 receptor agonist, but not a D_2 receptor agonist, mimicked the effects of progesterone in facilitating sexual behavior in female rats. The facilitatory effect of the neurotransmitter was blocked by progesterone receptor antagonists, a D_1 receptor antagonist, or antisense oligonucleotides to the progesterone receptor. The results suggest that in rodents neurotransmitters may regulate in vivo gene expression and behavior by means of cross-talk with steroid receptors in the brain.

Targeting GLP-1 receptor trafficking to improve agonist efficacy.

PubMed

Jones, Ben; Buenaventura, Teresa; Kanda, Nisha; Chabosseau, Pauline; Owen, Bryn M; Scott, Rebecca; Goldin, Robert; Angkathunyakul, Napat; Corrêa, Ivan R; Bosco, Domenico; Johnson, Paul R; Piemonti, Lorenzo; Marchetti, Piero; Shapiro, A M James; Cochran, Blake J; Hanyaloglu, Aylin C; Inoue, Asuka; Tan, Tricia; Rutter, Guy A; Tomas, Alejandra; Bloom, Stephen R

2018-04-23

Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in β-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.

The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

PubMed

Oakley, Robert H; Hudson, Christine C; Cruickshank, Rachael D; Meyers, Diane M; Payne, Richard E; Rhem, Shay M; Loomis, Carson R

2002-11-01

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

Prolonged morphine treatment alters δ opioid receptor post-internalization trafficking

PubMed Central

Ong, E W; Xue, L; Olmstead, M C; Cahill, C M

2015-01-01

BACKGROUND AND PURPOSE The δ opioid receptor (DOP receptor) undergoes internalization both constitutively and in response to agonists. Previous work has shown that DOP receptors traffic from intracellular compartments to neuronal cell membranes following prolonged morphine treatment. Here, we examined the effects of prolonged morphine treatment on the post-internalization trafficking of DOP receptors. EXPERIMENTAL APPROACH Using primary cultures of dorsal root ganglia neurons, we measured the co-localization of endogenous DOP receptors with post-endocytic compartments following both prolonged and acute agonist treatments. KEY RESULTS A departure from the constitutive trafficking pathway was observed following acute DOP receptor agonist-induced internalization by deltorphin II. That is, the DOP receptor underwent distinct agonist-induced post-endocytic sorting. Following prolonged morphine treatment, constitutive DOP receptor trafficking was augmented. SNC80 following prolonged morphine treatment also caused non-constitutive DOP receptor agonist-induced post-endocytic sorting. The μ opioid receptor (MOP receptor) agonist DAMGO induced DOP receptor internalization and trafficking following prolonged morphine treatment. Finally, all of the alterations to DOP receptor trafficking induced by both DOP and MOP receptor agonists were inhibited or absent when those agonists were co-administered with a DOP receptor antagonist, SDM-25N. CONCLUSIONS AND IMPLICATIONS The results support the hypothesis that prolonged morphine treatment induces the formation of MOP–DOP receptor interactions and subsequent augmentation of the available cell surface DOP receptors, at least some of which are in the form of a MOP/DOP receptor species. The pharmacology and trafficking of this species appear to be unique compared to those of its individual constituents. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24819092

Repeated stimulation of D1 dopamine receptors enhances (-)-11-hydroxy-delta 8-tetrahydrocannabinol-dimethyl-heptyl-induced catalepsy in male rats.

PubMed

Rodríguez de Fonseca, F; Martín Calderón, J L; Mechoulam, R; Navarro, M

1994-03-21

Dopaminergic and cannabinoid receptors are localized in the outflow nuclei of the basal ganglia. We have investigated the possible interrelation of these receptors in the regulation of motor activity in male rats. To this end we have first studied the behavioural effects of the highly potent cannabinoid receptor agonist (-)11-hydroxy-delta 8-tetrahydrocannabinol-dimethylheptyl (HU-210, 20 micrograms mg) after chronic stimulation of dopamine D1 and D2 receptors. The catalepsy induced by the synthetic cannabinoid, measured as the descent latency in the bar test, was enhanced in male rats chronically treated with the dopamine D1 receptor agonist SKF38393 (8 mg kg-1, twice a day during 21 days). However, animals exposed to the dopamine D2 agonist quinpirole (1 mg kg-1 daily during 21 days) displayed the same degree of catalepsy as those exposed to HU-210 alone. Although a possible involvement of D2 receptors cannot be excluded, this finding suggests a predominant role for dopamine D1 receptors in the regulation of the cataleptic response to cannabinoids. The possible cross-talk between dopamine D1 and cannabinoid receptors is further supported by the decreased responsiveness to the acute behavioural effects of SKF38393 (8 mg kg-1) observed in animals chronically exposed to HU-210 (20 micrograms kg-1 daily during 14 days).

Ligand-binding pocket shape differences between S1P1 and S1P3 determine efficiency of chemical probe identification by uHTS

PubMed Central

Schürer, Stephan C.; Brown, Steven J.; Cabrera, Pedro Gonzales; Schaeffer, Marie-Therese; Chapman, Jacqueline; Jo, Euijung; Chase, Peter; Spicer, Tim; Hodder, Peter; Rosen, Hugh

2008-01-01

We have studied the Sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 vs. S1P3 in this closely related receptor family. PMID:18590333

The Role of Dopamine in Reinforcement: Changes in Reinforcement Sensitivity Induced by D[subscript 1]-Type, D[subscript 2]-Type, and Nonselective Dopamine Receptor Agonists

ERIC Educational Resources Information Center

Bratcher, Natalie A.; Farmer-Dougan, Valeri; Dougan, James D.; Heidenreich, Byron A.; Garris, Paul A.

2005-01-01

Dose-dependent changes in sensitivity to reinforcement were found when rats were treated with low, moderate, and high doses of the partial dopamine D[subscript 1]-type receptor agonist SKF38393 and with the nonselective dopamine agonist apomorphine, but did not change when rats were treated with similar doses of the selective dopamine D[subscript…

Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells.

PubMed

Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Streitová, Denisa; Vacek, Antonín

2008-07-01

Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.

Bioassay-guided isolation of saikosaponins with agonistic activity on 5-hydroxytryptamine 2C receptor from Bupleurum chinense and their potential use for the treatment of obesity.

PubMed

Sun, Chang-Li; Geng, Chang-An; Huang, Xiao-Yan; Ma, Yun-Bao; Zheng, Xiao-Hong; Yang, Tong-Hua; Chen, Xing-Long; Yin, Xiu-Juan; Zhang, Xue-Mei; Chen, Ji-Jun

2017-06-01

5-Hydroxytryptamine 2C (5-HT 2C ) receptor is one of the major targets of anti-obesity agents, due to its role in regulation of appetite. In the present study, the 70% EtOH extract of the roots of Bupleurum chinense was revealed to have agonistic activity on 5-HT 2C receptor, and the subsequent bioassay-guided isolation led to identification of several saikosaponins as the active constituents with 5-HT 2C receptor agonistic activity in vitro and anti-obesity activity in vivo. The new compound, 22-oxosaikosaponin d (1), was determined by extensive spectroscopic analyses (HR-ESI-MS, IR, and 1D and 2D NMR). The primary structure-activity relationship study suggested that the intramolecular ether bond between C-13 and C-28 and the number of sugars at C-3 position were closely related to the 5-HT 2C receptor agonistic activity. Saikosaponin a (3), the main saponin in B. chinense, showed obviously agonistic activity on 5-HT 2C receptor with an EC 50 value of 21.08 ± 0.33 μmol·L -1 in vitro and could reduce food intake by 39.1% and 69.2%, and weight gain by 13.6% and 16.4%, respectively, at 3.0 and 6.0 mg·kg -1 in vivo. This investigation provided valuable information for the potential use of B. chinense as anti-obesity agent. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Differential profile of typical, atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: detection of agonist and inverse agonist properties.

PubMed

Rauly-Lestienne, Isabelle; Boutet-Robinet, Elisa; Ailhaud, Marie-Christine; Newman-Tancredi, Adrian; Cussac, Didier

2007-10-01

5-HT(7) receptors are present in thalamus and limbic structures, and a possible role of these receptors in the pathology of schizophrenia has been evoked. In this study, we examined binding affinity and agonist/antagonist/inverse agonist properties at these receptors of a large series of antipsychotics, i.e., typical, atypical, and third generation compounds preferentially targeting D(2) and 5-HT(1A) sites. Adenylyl cyclase (AC) activity was measured in HEK293 cells stably expressing the human (h) 5-HT(7a) receptor isoform. 5-HT and 5-CT increased cyclic adenosine monophosphate level by about 20-fold whereas (+)-8-OH-DPAT, the antidyskinetic agent sarizotan, and the novel antipsychotic compound bifeprunox exhibited partial agonist properties at h5-HT(7a) receptors stimulating AC. Other compounds antagonized 5-HT-induced AC activity with pK (B) values which correlated with their pK (i) as determined by competition binding vs [(3)H]5-CT. The selective 5-HT(7) receptor ligand, SB269970, was the most potent antagonist. For antipsychotic compounds, the following rank order of antagonism potency (pK (B)) was ziprasidone > tiospirone > SSR181507 > or = clozapine > or = olanzapine > SLV-314 > SLV-313 > or = aripiprazole > or = chlorpromazine > nemonapride > haloperidol. Interestingly, pretreatment of HEK293-h5-HT(7a) cells with forskolin enhanced basal AC activity and revealed inverse agonist properties for both typical and atypical antipsychotics as well as for aripiprazole. In contrast, other novel antipsychotics exhibited diverse 5-HT(7a) properties; SLV-313 and SLV-314 behaved as quasi-neutral antagonists, SSR181507 acted as an inverse agonist, and bifeprunox as a partial agonist, as mentioned above. In conclusion, the differential properties of third generation antipsychotics at 5-HT(7) receptors may influence their antipsychotic profile.

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway.

PubMed

Pende, A; Tremmel, K D; DeMaria, C T; Blaxall, B C; Minobe, W A; Sherman, J A; Bisognano, J D; Bristow, M R; Brewer, G; Port, J

1996-04-05

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells

PubMed Central

Shen, Wen; Slaughter, Malcolm M

1998-01-01

Glutamate suppressed high-voltage-activated barium currents (IBa,HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an ω-conotoxin GVIA-sensitive current. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (l-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized. PMID:9660896

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

PubMed

Elliott, P J; Mason, G S; Stephens-Smith, M; Hagan, R M

1991-06-01

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.

Evidence that the adenosine A3 receptor may mediate the protection afforded by preconditioning in the isolated rabbit heart.

PubMed

Liu, G S; Richards, S C; Olsson, R A; Mullane, K; Walsh, R S; Downey, J M

1994-07-01

Agonists selective for the A1 adenosine receptor mimic the protective effect of ischaemic preconditioning against infarction in the rabbit heart. Unselective adenosine antagonists block this protection but, paradoxically, the A1 adenosine receptor selective antagonist 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX) does not. The aim of this study was to test the hypothesis that the newly described A3 adenosine receptor, which has an agonist profile similar to the A1 receptor but is insensitive to DPCPX, might mediate preconditioning. Isolated rabbit hearts perfused with Krebs buffer experienced 30 min of regional ischaemia followed by 120 min of reperfusion. Infarct size was measured by tetrazolium staining. In control hearts infarction was 32.2(SEM 1.5)% of the risk zone. Preconditioning by 5 min ischaemia and 10 min reperfusion reduced infarct size to 8.8(2.3)%. Replacing the regional ischaemia with 5 min perfusion with 10 microM adenosine or 65 nM N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA), an adenosine A3 receptor agonist, was equally protective. The unselective antagonist 8-p-sulphophenyl theophylline at 100 microM abolished protection by preconditioning, adenosine, and APNEA, but 200 nM DPCPX did not block protection by any of the interventions. Likewise the potent but unselective A3 receptor antagonist 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BW A1433) completely blocked protection from ischaemic preconditioning. Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor.

Structure-5-HT/D2 Receptor Affinity Relationship in a New Group of 1-Arylpiperazynylalkyl Derivatives of 8-Dialkylamino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione.

PubMed

Żmudzki, Paweł; Satała, Grzegorz; Chłoń-Rzepa, Grażyna; Bojarski, Andrzej J; Kazek, Grzegorz; Siwek, Agata; Gryboś, Anna; Głuch-Lutwin, Monika; Wesołowska, Anna; Pawłowski, Maciej

2016-10-01

In our previous papers, we have reported that some 8-amino-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione derivatives possessed high affinity and displayed agonistic, partial agonistic, or antagonistic activity for serotonin 5-HT 1A and dopamine D 2 receptors. In order to examine further the influence of the substituent in the position 8 of the purine moiety and the influence of the xanthine core on the affinity for serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors, two series of 1-arylpiperazynylalkyl derivatives of 8-amino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione were synthesized. All the final compounds were investigated in in vitro competition binding experiments for the serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors. The structure-affinity relationships for this group of compounds were discussed. For selected compounds, the functional assays for the 5-HT 1A and D 2 receptors were carried out. The results of the assays indicated that these groups of derivatives possessed antagonistic activity for 5-HT 1A receptors and agonistic, partial agonistic, or antagonistic activity for D 2 receptors. In total, 26 new compounds were synthesized, 20 of which were tested in in vitro binding experiments and 5 were tested in in vitro functional assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relations between open-field, elevated plus-maze, and emergence tests in C57BL/6J and BALB/c mice injected with GABA- and 5HT-anxiolytic agents.

PubMed

Lalonde, Robert; Strazielle, Catherine

2010-06-01

Two 5HT(1A) receptor agonists and chlordiazepoxide were examined in open-field, elevated plus maze, and emergence tests. At doses with no effect in the open-field, chlordiazepoxide increased open and open/total arm visits as well as open arm duration in the elevated plus maze, whereas 5HT(1A) receptor agonists showed an anxiolytic response on a single measure. The anxiolytic action of chlordiazepoxide was limited to the less active BALB/c strain. Unlike the 5HT(1A) receptor agonists, chlordiazepoxide was also anxiolytic in the emergence test, once again only in BALB/c and not C57BL/6J mice. Significant correlations were found between emergence latencies and specific indicators of anxiety in the elevated plus-maze in chlordiazepoxide-treated but not in mice treated with buspirone and 8-OH-DPAT. These results indicate that elevated plus-maze and emergence tests depend on benzodiazepine receptors. In contrast, 5HT(1A) receptor agonists were ineffective in the emergence test and no correlation was found between emergence latencies and specific indicators of anxiety in the elevated plus-maze. Though superficially similar, the emergence test seems to tap into a partially separate facet of anxiety.

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

PubMed

Giuliani, S; Barbanti, G; Turini, D; Quartara, L; Rovero, P; Giachetti, A; Maggi, C A

1991-10-22

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration.

PubMed

Vaidya, Aditya S; Peterson, Francis C; Yarmolinsky, Dmitry; Merilo, Ebe; Verstraeten, Inge; Park, Sang-Youl; Elzinga, Dezi; Kaundal, Amita; Helander, Jonathan; Lozano-Juste, Jorge; Otani, Masato; Wu, Kevin; Jensen, Davin R; Kollist, Hannes; Volkman, Brian F; Cutler, Sean R

2017-11-17

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu; Cheng, Kunrong; Saxena, Neeraj

2011-11-18

Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasionmore » of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.« less

Differences in acute anorectic effects of long-acting GLP-1 receptor agonists in rats

USDA-ARS?s Scientific Manuscript database

Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists have both glucose- and weight-lowering effects. The brain is poised to mediate both of these actions since GLP-1Rs are present in key areas known to control weight and glucose. Although some research has been performed on the effects of ...

A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

PubMed Central

Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

2013-01-01

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

Neurochemical binding profiles of novel indole and benzofuran MDMA analogues.

PubMed

Shimshoni, Jakob A; Winkler, Ilan; Golan, Ezekiel; Nutt, David

2017-01-01

3,4-Methylenedioxy-N-methylamphetamine (MDMA) has been shown to be effective in the treatment of post-traumatic stress disorder (PTSD) in numerous clinical trials. In the present study, we have characterized the neurochemical binding profiles of three MDMA-benzofuran analogues (1-(benzofuran-5-yl)-propan-2-amine, 5-APB; 1-(benzofuran-6-yl)-N-methylpropan-2-amine, 6-MAPB; 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 5-MAPB) and one MDMA-indole analogue (1-(1H-indol-5-yl)-2-methylamino-propan-1-ol, 5-IT). These compounds were screened as potential second-generation anti-PTSD drugs, against a battery of human and non-human receptors, transporters, and enzymes, and their potencies as 5-HT 2 receptor agonist and monoamine uptake inhibitors determined. All MDMA analogues displayed high binding affinities for 5-HT 2a,b,c and NE α2 receptors, as well as significant 5-HT, DA, and NE uptake inhibition. 5-APB revealed significant agonist activity at the 5-HT 2a,b,c receptors, while 6-MAPB, 5-MAPB, and 5-IT exhibited significant agonist activity at the 5-HT 2c receptor. There was a lack of correlation between the results of functional uptake and the monoamine transporter binding assay. MDMA analogues emerged as potent and selective monoamine oxidase A inhibitors. Based on 6-MAPB favorable pharmacological profile, it was further subjected to IC 50 determination for monoamine transporters. Overall, all MDMA analogues displayed higher monoamine receptor/transporter binding affinities and agonist activity at the 5-HT 2a,c receptors as compared to MDMA.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.

PubMed

Lin, Alan L; Zhu, Bing; Zhang, WanKe; Dang, Howard; Zhang, Bin-Xian; Katz, Michael S; Yeh, Chih-Ko

2008-06-01

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.

TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy

DOE PAGES

Lim, So-Yon; Osuna, Christa E.; Hraber, Peter T.; ...

2018-05-02

Antiretroviral therapy can halt HIV-1 replication, but fails to target the long-lived latent viral reservoir. Several pharmacological compounds have been evaluated for their ability to reverse HIV-1 latency, but none have demonstrably reduced the latent HIV-1 reservoir, or impacted viral rebound following the interruption of antiretroviral therapy. Here, we evaluate orally administered selective tolllike receptor 7 agonists GS-986 and GS-9620 for their ability to induce transient viremia in simian immunodeficiency virus-infected rhesus monkeys on suppressive antiretroviral therapy. In an initial doseescalation study, and a subsequent dose-optimization study, we found that toll-like receptor 7 agonists activate multiple innate and adaptive immunemore » cell populations in addition to inducing SIV RNA. We also observed toll-like receptor 7 agonist-induced reductions in SIV DNA and ex vivo inducible virus from treated animals. In a second study, after stopping antiretroviral therapy, two of nine treated animals have remained aviremic for more than two years, even after in vivo CD8+ lymphocyte depletion. Moreover, adoptive transfer of cells from aviremic animals could not induce de novo infection in naive recipient macaques. These findings suggest that toll-like receptor agonists may facilitate reservoir reduction in a subset of individuals.« less

TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, So-Yon; Osuna, Christa E.; Hraber, Peter T.

Antiretroviral therapy can halt HIV-1 replication, but fails to target the long-lived latent viral reservoir. Several pharmacological compounds have been evaluated for their ability to reverse HIV-1 latency, but none have demonstrably reduced the latent HIV-1 reservoir, or impacted viral rebound following the interruption of antiretroviral therapy. Here, we evaluate orally administered selective tolllike receptor 7 agonists GS-986 and GS-9620 for their ability to induce transient viremia in simian immunodeficiency virus-infected rhesus monkeys on suppressive antiretroviral therapy. In an initial doseescalation study, and a subsequent dose-optimization study, we found that toll-like receptor 7 agonists activate multiple innate and adaptive immunemore » cell populations in addition to inducing SIV RNA. We also observed toll-like receptor 7 agonist-induced reductions in SIV DNA and ex vivo inducible virus from treated animals. In a second study, after stopping antiretroviral therapy, two of nine treated animals have remained aviremic for more than two years, even after in vivo CD8+ lymphocyte depletion. Moreover, adoptive transfer of cells from aviremic animals could not induce de novo infection in naive recipient macaques. These findings suggest that toll-like receptor agonists may facilitate reservoir reduction in a subset of individuals.« less

Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor

PubMed Central

Troppmann, B; Balfanz, S; Baumann, A; Blenau, W

2010-01-01

Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects. PMID:20233210

Use of CRISPR/Cas9-engineered INS-1 pancreatic β cells to define the pharmacology of dual GIPR/GLP-1R agonists.

PubMed

Naylor, Jacqueline; Suckow, Arthur T; Seth, Asha; Baker, David J; Sermadiras, Isabelle; Ravn, Peter; Howes, Rob; Li, Jianliang; Snaith, Mike R; Coghlan, Matthew P; Hornigold, David C

2016-09-15

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

PubMed

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Investigation of pyrazolo-sulfonamides as putative small molecule oxytocin receptor agonists.

PubMed

Katte, Timothy A; Reekie, Tristan A; Werry, Eryn L; Jorgensen, William T; Boyd, Rochelle; Wong, Erick C N; Gulliver, Damien W; Connor, Mark; Kassiou, Michael

2017-08-18

The neuropeptide oxytocin has been implicated in multiple central nervous system functions in mammalian species. Increased levels have been reported to improve trust, alleviate symptoms related to autism and social phobias, and reduce social anxiety. Hoffman-La Roche published a patent claiming to have found potent small molecule oxytocin receptor agonists, smaller than the first non-peptide oxytocin agonist reported, WAY 267,464. We selected two of the more potent compounds from the patent and, in addition, created WAY 267,464 hybrid structures and determined their oxytocin and vasopressin receptor activity. Human embryonic kidney and Chinese hamster ovary cells were used for the expression of oxytocin or vasopressin 1a receptors and activity assessed via IP1 accumulation assays and calcium FLIPR assays. The results concluded that the reported compounds in the patent and the hybrid structures have no activity at the oxytocin or vasopressin 1a receptors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms.

PubMed

Carlin, Jesse Lea; Jain, Shalini; Gizewski, Elizabeth; Wan, Tina C; Tosh, Dilip K; Xiao, Cuiying; Auchampach, John A; Jacobson, Kenneth A; Gavrilova, Oksana; Reitman, Marc L

2017-03-01

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1 -/- , Adora3 -/- ) mice. Confirming prior data, stimulation of the A 3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H 1 receptors. In contrast, A 1 AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A 1 AR agonists, including N 6 -cyclopentyladenosine (CPA), N 6 -cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A 1 AR and A 3 AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N 6 -endo-norbornyladenosine], or using Adora3 -/- mice allowed selective stimulation of A 1 AR. AMP-stimulated hypothermia can occur independently of A 1 AR, A 3 AR, and mast cells. A 1 AR and A 3 AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A 1 AR nor A 3 AR was required for fasting-induced torpor. A 1 AR and A 3 AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms. Published by Elsevier Ltd.

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

PubMed

Zwart, R; Abraham, D; Oortgiesen, M; Vijverberg, H P

1994-08-22

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine > cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon

PubMed Central

Warner, Fiona J; Comis, Alfio; Miller, Robert C; Burcher, Elizabeth

1999-01-01

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (KD 0.47±0.05 nM), of high capacity (Bmax 2.1±0.1 fmol mg−1 wet weight tissue) and reversible (kinetically derived KD 0.36±0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide γ (NPγ)≥NKA≥[Lys5,MeLeu9,Nle10]NKA (4–10) (NK2 agonist)>>substance P (SP)>neurokinin B (NKB)≥[Pro9]SP (NK1 agonist)>>senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968≥MEN11420>GR94800≥MEN10627>MEN10376≥R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 μM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear. PMID:10455255

Systematic review: cardiovascular safety profile of 5-HT(4) agonists developed for gastrointestinal disorders.

PubMed

Tack, J; Camilleri, M; Chang, L; Chey, W D; Galligan, J J; Lacy, B E; Müller-Lissner, S; Quigley, E M M; Schuurkes, J; De Maeyer, J H; Stanghellini, V

2012-04-01

The nonselective 5-HT(4) receptor agonists, cisapride and tegaserod have been associated with cardiovascular adverse events (AEs). To perform a systematic review of the safety profile, particularly cardiovascular, of 5-HT(4) agonists developed for gastrointestinal disorders, and a nonsystematic summary of their pharmacology and clinical efficacy. Articles reporting data on cisapride, clebopride, prucalopride, mosapride, renzapride, tegaserod, TD-5108 (velusetrag) and ATI-7505 (naronapride) were identified through a systematic search of the Cochrane Library, Medline, Embase and Toxfile. Abstracts from UEGW 2006-2008 and DDW 2008-2010 were searched for these drug names, and pharmaceutical companies approached to provide unpublished data. Retrieved articles on pharmacokinetics, human pharmacodynamics and clinical data with these 5-HT(4) agonists, are reviewed and summarised nonsystematically. Articles relating to cardiac safety and tolerability of these agents, including any relevant case reports, are reported systematically. Two nonselective 5-HT(4) agonists had reports of cardiovascular AEs: cisapride (QT prolongation) and tegaserod (ischaemia). Interactions with, respectively, the hERG cardiac potassium channel and 5-HT(1) receptor subtypes have been suggested to account for these effects. No cardiovascular safety concerns were reported for the newer, selective 5-HT(4) agonists prucalopride, velusetrag, naronapride, or for nonselective 5-HT(4) agonists with no hERG or 5-HT(1) affinity (renzapride, clebopride, mosapride). 5-HT(4) agonists for GI disorders differ in chemical structure and selectivity for 5-HT(4) receptors. Selectivity for 5-HT(4) over non-5-HT(4) receptors may influence the agent's safety and overall risk-benefit profile. Based on available evidence, highly selective 5-HT(4) agonists may offer improved safety to treat patients with impaired GI motility. © 2012 Blackwell Publishing Ltd.

Systematic review: cardiovascular safety profile of 5-HT4 agonists developed for gastrointestinal disorders

PubMed Central

Tack, J; Camilleri, M; Chang, L; Chey, W D; Galligan, J J; Lacy, B E; Müller-Lissner, S; Quigley, E M M; Schuurkes, J; Maeyer, J H; Stanghellini, V

2012-01-01

Summary Background The nonselective 5-HT4 receptor agonists, cisapride and tegaserod have been associated with cardiovascular adverse events (AEs). Aim To perform a systematic review of the safety profile, particularly cardiovascular, of 5-HT4 agonists developed for gastrointestinal disorders, and a nonsystematic summary of their pharmacology and clinical efficacy. Methods Articles reporting data on cisapride, clebopride, prucalopride, mosapride, renzapride, tegaserod, TD-5108 (velusetrag) and ATI-7505 (naronapride) were identified through a systematic search of the Cochrane Library, Medline, Embase and Toxfile. Abstracts from UEGW 2006–2008 and DDW 2008–2010 were searched for these drug names, and pharmaceutical companies approached to provide unpublished data. Results Retrieved articles on pharmacokinetics, human pharmacodynamics and clinical data with these 5-HT4 agonists, are reviewed and summarised nonsystematically. Articles relating to cardiac safety and tolerability of these agents, including any relevant case reports, are reported systematically. Two nonselective 5-HT4 agonists had reports of cardiovascular AEs: cisapride (QT prolongation) and tegaserod (ischaemia). Interactions with, respectively, the hERG cardiac potassium channel and 5-HT1 receptor subtypes have been suggested to account for these effects. No cardiovascular safety concerns were reported for the newer, selective 5-HT4 agonists prucalopride, velusetrag, naronapride, or for nonselective 5-HT4 agonists with no hERG or 5-HT1 affinity (renzapride, clebopride, mosapride). Conclusions 5-HT4 agonists for GI disorders differ in chemical structure and selectivity for 5-HT4 receptors. Selectivity for 5-HT4 over non-5-HT4 receptors may influence the agent's safety and overall risk–benefit profile. Based on available evidence, highly selective 5-HT4 agonists may offer improved safety to treat patients with impaired GI motility. PMID:22356640

Novel selective agonists and antagonists confirm neurokinin NK1 receptors in guinea-pig vas deferens.

PubMed Central

Hall, J. M.; Morton, I. K.

1991-01-01

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707714

Cellular and synaptic effect of substance P on neonatal phrenic motoneurons.

PubMed

Ptak, K; Konrad, M; Di Pasquale, E; Tell, F; Hilaire, G; Monteau, R

2000-01-01

Experiments were carried out on the in vitro brainstem-spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current-clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency-intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP-mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin-containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage-clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4-aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M-type potassium current but only a current showing no voltage sensitivity over the range -100 to 0 mV, reversing near the expected EK +, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.

Identification and mechanism of ABA receptor antagonism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melcher, Karsten; Xu, Yong; Ng, Ley-Moy

2010-11-11

The phytohormone abscisic acid (ABA) functions through a family of fourteen PYR/PYL receptors, which were identified by resistance to pyrabactin, a synthetic inhibitor of seed germination. ABA activates these receptors to inhibit type 2C protein phosphatases, such as ABI1, yet it remains unclear whether these receptors can be antagonized. Here we demonstrate that pyrabactin is an agonist of PYR1 and PYL1 but is unexpectedly an antagonist of PYL2. Crystal structures of the PYL2-pyrabactin and PYL1-pyrabactin-ABI1 complexes reveal the mechanism responsible for receptor-selective activation and inhibition, which enables us to design mutations that convert PYL1 to a pyrabactin-inhibited receptor and PYL2more » to a pyrabactin-activated receptor and to identify new pyrabactin-based ABA receptor agonists. Together, our results establish a new concept of ABA receptor antagonism, illustrate its underlying mechanisms and provide a rational framework for discovering novel ABA receptor ligands.« less

Bitter taste receptor agonists alter mitochondrial function and induce autophagy in airway smooth muscle cells.

PubMed

Pan, Shi; Sharma, Pawan; Shah, Sushrut D; Deshpande, Deepak A

2017-07-01

Airway remodeling, including increased airway smooth muscle (ASM) mass, is a hallmark feature of asthma and COPD. We previously identified the expression of bitter taste receptors (TAS2Rs) on human ASM cells and demonstrated that known TAS2R agonists could promote ASM relaxation and bronchodilation and inhibit mitogen-induced ASM growth. In this study, we explored cellular mechanisms mediating the antimitogenic effect of TAS2R agonists on human ASM cells. Pretreatment of ASM cells with TAS2R agonists chloroquine and quinine resulted in inhibition of cell survival, which was largely reversed by bafilomycin A1, an autophagy inhibitor. Transmission electron microscope studies demonstrated the presence of double-membrane autophagosomes and deformed mitochondria. In ASM cells, TAS2R agonists decreased mitochondrial membrane potential and increased mitochondrial ROS and mitochondrial fragmentation. Inhibiting dynamin-like protein 1 (DLP1) reversed TAS2R agonist-induced mitochondrial membrane potential change and attenuated mitochondrial fragmentation and cell death. Furthermore, the expression of mitochondrial protein BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) and mitochondrial localization of DLP1 were significantly upregulated by TAS2R agonists. More importantly, inhibiting Bnip3 mitochondrial localization by dominant-negative Bnip3 significantly attenuated cell death induced by TAS2R agonist. Collectively the TAS2R agonists chloroquine and quinine modulate mitochondrial structure and function, resulting in ASM cell death. Furthermore, Bnip3 plays a central role in TAS2R agonist-induced ASM functional changes via a mitochondrial pathway. These findings further establish the cellular mechanisms of antimitogenic effects of TAS2R agonists and identify a novel class of receptors and pathways that can be targeted to mitigate airway remodeling as well as bronchoconstriction in obstructive airway diseases. Copyright © 2017 the American Physiological Society.

1,2,3-Triazole Rings as a Disulfide Bond Mimetic in Chimeric AGRP-Melanocortin Peptides: Design, Synthesis, and Functional Characterization.

PubMed

Tala, Srinivasa R; Singh, Anamika; Lensing, Cody J; Schnell, Sathya M; Freeman, Katie T; Rocca, James R; Haskell-Luevano, Carrie

2018-05-16

The melanocortin system is involved in the regulation of complex physiological functions, including energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. The five melanocortin receptors that belong to the superfamily of G protein-coupled receptors (GPCRs) are regulated by endogenously expressed agonists and antagonists. The aim of this study was to explore the potential of replacing the disulfide bridge in chimeric AGRP-melanocortin peptide Tyr-c[Cys-His-d-Phe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH 2 (1) with 1,2,3-triazole moieties. A series of 1,2,3-triazole-bridged peptidomimetics were designed, synthesized, and pharmacologically evaluated at the mouse melanocortin receptors. The ligands possessed nanomolar to micromolar agonist cAMP signaling potency. A key finding was that the disulfide bond in peptide 1 can be replaced with the monotriazole ring with minimal effect on the functional activity at the melanocortin receptors. The 1,5-disubstituted triazole-bridged peptide 6 showed equipotent functional activity at the mMC3R and modest 5-fold decreased agonist potency at the mMC4R compared to those of 1. Interestingly, the 1,4- and 1,5-disubstituted isomers of the triazole ring resulted in different selectivities at the receptor subtypes, indicating subtle structural features that may be exploited in the generation of selective melanocortin ligands. Introducing cyclic and acyclic bis-triazole moieties into chimeric AGRP template 1 generally decreased agonist activity. These results will be useful for the further design of neuronal chemical probes for the melanocortin receptors as well as in other receptor systems.

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands.

PubMed

Bender, Aaron M; Clark, Mary J; Agius, Michael P; Traynor, John R; Mosberg, Henry I

2014-01-15

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

PubMed

Newman-Tancredi, Adrian; Assié, Marie-Bernadette; Leduc, Nathalie; Ormière, Anne-Marie; Danty, Nathalie; Cosi, Cristina

2005-09-01

Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the antipsychotics will depend on the relative 5-HT1A/D2 affinities and on 5-HT1A agonist efficacy of the drugs.

Ethanol Inhibition of Constitutively Open N-Methyl-d-Aspartate Receptors

PubMed Central

Xu, Minfu; Smothers, C. Thetford; Trudell, James

2012-01-01

N-Methyl-d-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits. PMID:22005043

The relationship between reinforcing effects and in vitro effects of D1 agonists in monkeys.

PubMed

Weed, M R; Paul, I A; Dwoskin, L P; Moore, S E; Woolverton, W L

1997-10-01

The reinforcing effects of many psychomotor stimulants have been related to increased dopaminergic neurotransmission and stimulation of central nervous system (CNS) dopamine (DA) receptors. Consistent with this notion, some drugs that directly stimulate DA receptors have been found to function as positive reinforcers. The present experiments were designed to examine why some, but not all, D1 receptor agonists can function as reinforcers in rhesus monkeys by comparing behavioral and CNS in vitro measures of potency and efficacy. Seven rhesus monkeys were allowed to self-administer cocaine under a progressive-ratio (PR) schedule until stable responding was established. Various doses of D1 agonists, previously reported to function as positive reinforcers, were then made available for self-administration. Stimulation of cAMP production in rhesus and rat striatal tissue was studied for these compounds and for D1 agonists previously reported not to function as positive reinforcers in monkeys (SKF 38393, SKF 77434 and S(-)-6-BrAPB). Blockade of DA uptake in rat striata was also examined for all compounds. SKF 81297, SKF 82958 and R(+)-6-BrAPB maintained responding under the PR schedule and did not differ significantly in efficacy as positive reinforcers; SKF 81297 was less potent than the other two agonists. SKF 81297, SKF 82958 and R(+)-6-BrAPB stimulated higher levels of cAMP production in rhesus striata than did SKF 38393, SKF 77434 and S(-)-6-BrAPB. In contrast, all compounds blocked DA uptake. Thus, reinforcing efficacy among D1 agonists increases with efficacy in stimulating D1 receptors.

Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function

PubMed Central

Perroy, Julie; Walwyn, Wendy M.; Smith, Monique L.; Vicente-Sanchez, Ana; Segura, Laura; Bana, Alia; Kieffer, Brigitte L.; Evans, Christopher J.

2016-01-01

Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor–Ca2+ channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization. PMID:27013682

Selective CB2 receptor agonists. Part 2: Structure-activity relationship studies and optimization of proline-based compounds.

PubMed

Riether, Doris; Zindell, Renee; Wu, Lifen; Betageri, Raj; Jenkins, James E; Khor, Someina; Berry, Angela K; Hickey, Eugene R; Ermann, Monika; Albrecht, Claudia; Ceci, Angelo; Gemkow, Mark J; Nagaraja, Nelamangala V; Romig, Helmut; Sauer, Achim; Thomson, David S

2015-02-01

Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Membrane Potential Controls the Efficacy of Catecholamine-induced β1-Adrenoceptor Activity*

PubMed Central

Birk, Alexandra; Rinne, Andreas; Bünemann, Moritz

2015-01-01

G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (VM) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled β1-adrenoreceptor (β1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by VM. This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of β2-AR activation, however, was weak compared with β1-AR voltage-dependence. Drug efficacy is a major target of β1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of VM and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied β1-adrenoceptors on a very fast time scale. PMID:26408198

Vasopressin-induced constriction of the isolated rat occipital artery is segment dependent.

PubMed

Chelko, Stephen P; Schmiedt, Chad W; Lewis, Tristan H; Lewis, Stephen J; Robertson, Tom P

2013-01-01

Circulating factors delivered to the nodose ganglion (NG) by the occipital artery (OA) have been shown to affect vagal afferent activity, and thus the contractile state of the OA may influence blood flow to the NG. OA were isolated and bisected into proximal and distal segments relative to the external carotid artery. Bisection highlighted stark differences between maximal contractile responses and OA sensitivity. Specifically, maximum responses to vasopressin and the V1 receptor agonist were significantly higher in distal than proximal segments. Distal segments were significantly more sensitive to 5-hydroxytryptamine (5-HT) and the 5-HT2 receptor agonist than proximal segments. Angiotensin II (AT)2, V2 and 5-HT(1B/1D) receptor agonists did not elicit vascular responses. Additionally, AT1 receptor agonists elicited mild, yet not significantly different maximal responses between segments. The results of this study are consistent with contractile properties of rat OA being mediated via AT1, V1 and 5-HT2 receptors and dependent upon the OA segment. Furthermore, vasopressin-induced constriction of the OA, regardless of a bolus dose or a first and second concentration-response curve, retained this unique segmental difference. We hypothesize that these segmental differences may be important in the regulation of blood flow through the OA in health and disease. © 2013 S. Karger AG, Basel.

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents.

PubMed

Fox, Norma E; Lim, Jihyang; Chen, Rose; Geddis, Amy E

2010-05-01

To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists. We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species. Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin. These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin. 2010 ISEH Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

When Intensive Insulin Therapy (MDI) Fails in Patients With Type 2 Diabetes: Switching to GLP-1 Receptor Agonist Versus Insulin Pump.

PubMed

Cohen, Ohad; Filetti, Sebastiano; Castañeda, Javier; Maranghi, Marianna; Glandt, Mariela

2016-08-01

Treatment with insulin, alone or with oral or injectable hypoglycemic agents, is becoming increasingly common in patients with type 2 diabetes. However, approximately 40% of patients fail to reach their glycemic targets with the initially prescribed regimen and require intensification of insulin therapy, which increases the risks of weight gain and hypoglycemia. Many of these patients eventually reach a state in which further increases in the insulin dosage fail to improve glycemic control while increasing the risks of weight gain and hypoglycemia. The recently completed OpT2mise clinical trial showed that continuous subcutaneous insulin infusion (CSII) is more effective in reducing glycated hemoglobin (HbA1c) than intensification of multiple daily injection (MDI) insulin therapy in patients with type 2 diabetes who do not respond to intensive insulin therapy. CSII therapy may also be useful in patients who do not reach glycemic targets despite multidrug therapy with basal-bolus insulin and other agents, including glucagon-like peptide (GLP)-1 receptor agonists; current guidelines offer no recommendations for the treatment of such patients. Importantly, insulin and GLP-1 receptor agonists have complementary effects on glycemia and, hence, can be used either sequentially or in combination in the initial management of diabetes. Patients who have not previously failed GLP-1 receptor agonist therapy may show reduction in weight and insulin dose, in addition to moderate improvement in HbA1c, when GLP-1 receptor agonist therapy is added to MDI regimens. In subjects with long-standing type 2 diabetes who do not respond to intensive insulin therapies, switching from MDI to CSII and/or the addition of GLP-1 receptor agonists to MDI have the potential to improve glycemic control without increasing the risk of adverse events. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding.

PubMed Central

Negri, L.; Improta, G.; Lattanzi, R.; Potenza, R. L.; Luchetti, F.; Melchiorri, P.

1995-01-01

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist. PMID:8680727

Prolonged morphine treatment alters δ opioid receptor post-internalization trafficking.

PubMed

Ong, E W; Xue, L; Olmstead, M C; Cahill, C M

2015-01-01

The δ opioid receptor (DOP receptor) undergoes internalization both constitutively and in response to agonists. Previous work has shown that DOP receptors traffic from intracellular compartments to neuronal cell membranes following prolonged morphine treatment. Here, we examined the effects of prolonged morphine treatment on the post-internalization trafficking of DOP receptors. Using primary cultures of dorsal root ganglia neurons, we measured the co-localization of endogenous DOP receptors with post-endocytic compartments following both prolonged and acute agonist treatments. A departure from the constitutive trafficking pathway was observed following acute DOP receptor agonist-induced internalization by deltorphin II. That is, the DOP receptor underwent distinct agonist-induced post-endocytic sorting. Following prolonged morphine treatment, constitutive DOP receptor trafficking was augmented. SNC80 following prolonged morphine treatment also caused non-constitutive DOP receptor agonist-induced post-endocytic sorting. The μ opioid receptor (MOP receptor) agonist DAMGO induced DOP receptor internalization and trafficking following prolonged morphine treatment. Finally, all of the alterations to DOP receptor trafficking induced by both DOP and MOP receptor agonists were inhibited or absent when those agonists were co-administered with a DOP receptor antagonist, SDM-25N. The results support the hypothesis that prolonged morphine treatment induces the formation of MOP-DOP receptor interactions and subsequent augmentation of the available cell surface DOP receptors, at least some of which are in the form of a MOP/DOP receptor species. The pharmacology and trafficking of this species appear to be unique compared to those of its individual constituents. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

PubMed Central

Jacobson, Kenneth A.; Constanzi, Stefano; Ohno, Michihiro; Joshi, Bhalchandra V.; Besada, Pedro; Xu, Bin; Tchilibon, Susanna

2015-01-01

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7. PMID:15078212

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064.

PubMed

Akwabi-Ameyaw, Adwoa; Bass, Jonathan Y; Caldwell, Richard D; Caravella, Justin A; Chen, Lihong; Creech, Katrina L; Deaton, David N; Jones, Stacey A; Kaldor, Istvan; Liu, Yaping; Madauss, Kevin P; Marr, Harry B; McFadyen, Robert B; Miller, Aaron B; Navas, Frank; Parks, Derek J; Spearing, Paul K; Todd, Dan; Williams, Shawn P; Wisely, G Bruce

2008-08-01

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

PubMed

Yanofsky, Stephen D; Shen, Emily S; Holden, Frank; Whitehorn, Erik; Aguilar, Barbara; Tate, Emily; Holmes, Christopher P; Scheuerman, Randall; MacLean, Derek; Wu, May M; Frail, Donald E; López, Francisco J; Winneker, Richard; Arey, Brian J; Barrett, Ronald W

2006-05-12

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Histaminergic H3-Heteroreceptors as a Potential Mediator of Betahistine-Induced Increase in Cochlear Blood Flow.

PubMed

Bertlich, Mattis; Ihler, Friedrich; Freytag, Saskia; Weiss, Bernhard G; Strupp, Michael; Canis, Martin

2015-01-01

Betahistine is a histamine-like drug that is considered beneficial in Ménière's disease by increasing cochlear blood flow. Acting as an agonist at the histamine H1-receptor and as an inverse agonist at the H3-receptor, these receptors as well as the adrenergic α2-receptor were investigated for betahistine effects on cochlear blood flow. A total of 54 Dunkin-Hartley guinea pigs were randomly assigned to one of nine groups treated with a selection of H1-, H3- or α2-selective agonists and antagonists together with betahistine. Cochlear blood flow and mean arterial pressure were recorded for 3 min before and 15 min after infusion. Blockage of the H3- or α2-receptors caused a suppression of betahistine-mediated typical changes in cochlear blood flow or blood pressure. Activation of H3-receptors caused a drop in cochlear blood flow and blood pressure. H1-receptors showed no involvement in betahistine-mediated changes of cochlear blood flow. Betahistine most likely affects cochlear blood flow through histaminergic H3-heteroreceptors. © 2015 S. Karger AG, Basel.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids*

PubMed Central

Anavi-Goffer, Sharon; Baillie, Gemma; Irving, Andrew J.; Gertsch, Jürg; Greig, Iain R.; Pertwee, Roger G.; Ross, Ruth A.

2012-01-01

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55. PMID:22027819

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants

PubMed Central

Wifling, D; Löffel, K; Nordemann, U; Strasser, A; Bernhardt, G; Dove, S; Seifert, R; Buschauer, A

2015-01-01

Background and Purpose Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4R) and rat H4 receptor (rH4R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M; double mutants: hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S. Experimental Approach Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([3H]-histamine), and in functional [35S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Key Results Constitutive activity decreased from the hH4 receptor via the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. Conclusions and Implications F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc PMID:24903527

Involvement of the BLT2 receptor in the itch-associated scratching induced by 12-(S)-lipoxygenase products in ICR mice

PubMed Central

Kim, H J; Kim, D K; Kim, H; Koh, J Y; Kim, K M; Noh, M S; Lee, S; Kim, S; Park, S H; Kim, J J; Kim, S Y; Lee, C H

2008-01-01

Background and purpose: Recently, we reported that 12(S)-HPETE (12(S)-hydroperoxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid) induces scratching in ICR mice. We hypothesized that 12(S)-HPETE might act as an agonist of the low-affinity leukotriene B4 receptor BLT2. To confirm the involvement of the BLT2 receptor in 12(S)-HPETE-induced scratching, we studied the scratch response using the BLT2 receptor agonists compound A (4′-{[pentanoyl (phenyl) amino]methyl}-1,1′-biphenyl-2-carboxylic acid) and 12(S)-HETE (12(S)-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid). Experimental approach: A video recording was used to determine whether the BLT2 receptor agonists caused itch-associated scratching in ICR mice. Selective antagonists and several chemicals were used. Key results: Both 12(S)-HETE and compound A dose dependently induced scratching in the ICR mice. The dose–response curve for compound A showed peaks at around 0.005–0.015 nmol per site. Compound A- and 12(S)-HETE-induced scratching was suppressed by capsaicin and naltrexon. We examined the suppressive effects of U75302 (6-[6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl]-1,5-hexanediol, the BLT1 receptor antagonist) and LY255283 (1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]phenyl]-ethanone, the BLT2 receptor antagonist) on the BLT2 agonist-induced scratching. LY255283 suppressed compound A- and 12(S)-HETE-induced scratching, but U75302 did not. LY255283 required a higher dose to suppress the compound A-induced scratching than it did to suppress the 12(S)-HETE-induced scratching. One of the BLT2 receptor agonists, 12(R)-HETE (12(R)-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid), also induced scratching in the ICR mice. Conclusions and implications: Our present results corroborate the hypothesis that the BLT2 receptor is involved in 12(S)-lipoxygenase-product-induced scratching in ICR mice. We also confirmed that this animal model could be a valuable means of evaluating the effects of BLT2 receptor antagonists. PMID:18536755

Structural prerequisites for G-protein activation by the neurotensin receptor

DOE PAGES

Krumm, Brian E.; White, Jim F.; Shah, Priyanka; ...

2015-07-24

We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

Structural prerequisites for G-protein activation by the neurotensin receptor

PubMed Central

Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

2015-01-01

We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

Structural prerequisites for G-protein activation by the neurotensin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krumm, Brian E.; White, Jim F.; Shah, Priyanka

We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2).

PubMed

Fotsch, Christopher; Smith, Duncan M; Adams, Jeffrey A; Cheetham, Janet; Croghan, Michael; Doherty, Elizabeth M; Hale, Clarence; Jarosinski, Mark A; Kelly, Michael G; Norman, Mark H; Tamayo, Nuria A; Xi, Ning; Baumgartner, James W

2003-07-21

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.

Discovery of novel acetanilide derivatives as potent and selective beta3-adrenergic receptor agonists.

PubMed

Maruyama, Tatsuya; Onda, Kenichi; Hayakawa, Masahiko; Matsui, Tetsuo; Takasu, Toshiyuki; Ohta, Mitsuaki

2009-06-01

In the search for potent and selective human beta3-adrenergic receptor (AR) agonists as potential drugs for the treatment of obesity and noninsulin-dependent (type II) diabetes, a novel series of acetanilide-based analogues were prepared and their biological activities were evaluated at the human beta3-, beta2-, and beta1-ARs. Among these compounds, 2-pyridylacetanilide (2f), pyrimidin-2-ylacetanilide (2u), and pyrazin-2-ylacetanilide (2v) derivatives exhibited potent agonistic activity at the beta3-AR with functional selectivity over the beta1- and beta2-ARs. In particular, compound 2u was found to be the most potent and selective beta3-AR agonist with an EC(50) value of 0.11 microM and no agonistic activity for either the beta1- or beta2-AR. In addition, 2f, 2u, and 2v showed significant hypoglycemic activity in a rodent diabetic model.

Effect of the α2 -receptor agonists medetomidine, detomidine, xylazine, and romifidine on the ketamine metabolism in equines assessed with enantioselective capillary electrophoresis.

PubMed

Sandbaumhüter, Friederike A; Theurillat, Regula; Bettschart-Wolfensberger, Regula; Thormann, Wolfgang

2017-08-01

The combination of ketamine and an α 2 -receptor agonist is often used in veterinary medicine. Four different α 2 -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α 2 -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α 2 -receptor agonist is dependent on the α 2 -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (ii) the formation of the ketamine metabolites norketamine, 6-hydroxynorketamine (6HNK), and 5,6-dehydronorketamine (DHNK) in presence of the four α 2 -receptor agonists and equine liver microsomes. Samples were analyzed with enantioselective capillary electrophoresis using highly sulfated γ-cyclodextrin as chiral selector. All four α 2 -receptor agonists have an impact on the ketamine metabolism. Medetomidine was found to be the strongest inhibitor, followed by detomidine, whereas xylazine and romifidine showed almost no effect on the ketamine N-demethylation in the inhibition studies with a short-incubation period of the reaction mixture. After prolonged incubation, inhibition with xylazine and romifidine was also observed. The formation of 6HNK and DHNK is affected by all selected α 2 -receptor agonists. With medetomidine, levels of these metabolites are reduced compared to the case without an α 2 -receptor agonist. For detomidine, xylazine, and romifidine, the opposite was found. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display.

PubMed

Sakamoto, Kotaro; Kawata, Yayoi; Masuda, Yasushi; Umemoto, Tadashi; Ito, Takashi; Asami, Taiji; Takekawa, Shiro; Ohtaki, Tetsuya; Inooka, Hiroshi

2016-11-04

Fibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The involvement of medial septum 5-HT1 and 5-HT2 receptors on ACPA-induced memory consolidation deficit: possible role of TRPC3, TRPC6 and TRPV2.

PubMed

Najar, Farzaneh; Nasehi, Mohammad; Haeri-Rohani, Seyed-Ali; Zarrindast, Mohammad-Reza

2015-11-01

The present study evaluates the roles of serotonergic receptors of the medial septum on amnesia induced by arachidonylcyclopropylamide (ACPA; as selective cannabinoid CB1 receptor agonist) in adult male Wistar rats. Cannulae were implanted in the medial septum of the brain of the rats. The animals were trained in a passive avoidance learning apparatus, and were tested 24 hours after training for step-through latency. Results indicated that post-training medial septum administration of CP94253 (5-HT1B/1D receptor agonist) and cinancerine (as 5-HT2 receptor antagonist) reduced the step-through latency showing an amnesic response, while GR127935 (5-HT1B/1D receptor antagonist) and αm5htm (as 5-HT2A/2B/2D receptor agonist) did not alter memory consolidation by themselves. On continuing the test, the results showed that CP94253 increased and GR127935 did not alter ACPA (0.02 µg/rat)-induced memory impairment, respectively. Other data indicated that αm5htm induced a modulatory effect, while cinancerine restored ACPA-induced amnesia. Using SKF-96365 (inhibitor of transient receptor potential TRPC3/6 and TRPV2 channels) demonstrated that TRPC3, TRPC3 and TRPV2 channels have a significant role, according to our results. © The Author(s) 2015.

Cannabis and cannabinoids: pharmacology and rationale for clinical use.

PubMed

Pertwee, R G

1999-10-01

It is now known that there are at least two types of cannabinoid receptors. These are CB1 receptors, present mainly on central and peripheral neurones, and CB2 receptors, present mainly on immune cells. Endogenous cannabinoid receptor agonists ('endocannabinoids') have also been identified. The discovery of this 'endogenous cannabinoid system' has led to the development of selective CB1 and CB2 receptor ligands and fueled renewed interest in the clinical potential of cannabinoids. Two cannabinoid CB1 receptor agonists are already used clinically, as antiemetics or as appetite stimulants. These are D 9 - tetrahydrocannabinol (THC) and nabilone. Other possible uses for CB1 receptor agonists include the suppression of muscle spasm/spasticity associated with multiple sclerosis or spinal cord injury, the relief of chronic pain and the management of glaucoma and bronchial asthma. CB1 receptor antagonists may also have clinical applications, e. g. as appetite suppressants and in the management of schizophrenia or disorders of cognition and memory. So too may CB2 receptor ligands and drugs that activate cannabinoid receptors indirectly by augmenting endocannabinoid levels at cannabinoid receptors. When taken orally, THC seems to undergo variable absorption and to have a narrow 'therapeutic window' (dose range in which it is effective without producing significant unwanted effects). This makes it difficult to predict an oral dose that will be both effective and tolerable to a patient and indicates a need for better cannabinoid formulations and modes of administration. For the therapeutic potential of cannabis or CB1 receptor agonists to be fully exploited, it will be important to establish objectively and conclusively (a) whether these agents have efficacy against selected symptoms that is of clinical significance and, if so, whether the benefits outweigh the risks, (b) whether cannabis has therapeutic advantages over individual cannabinoids, (c) whether there is a need for additional drug treatments to manage any of the disorders against which cannabinoids are effective, and (d) whether it will be possible to develop drugs that have reduced psychotropic activity and yet retain the ability to act through CB1 receptors to produce their sought-after effects. Copyright 1999 S. Karger GmbH, Freiburg

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

PubMed

Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

PubMed Central

Gavina, Manuela; Za, Lorena; Molteni, Raffaella; Pardi, Ruggero; Curtis, Ivan de

2009-01-01

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins. PMID:19912111

Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

PubMed

Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

2017-08-15

Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

PubMed Central

Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

2012-01-01

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task.

PubMed

Perez-García, Georgina S; Meneses, A

2005-08-30

This work aimed to evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Post-training administration of the potential 5-HT7 receptor agonist AS 19 or antagonist SB-269970 enhanced memory formation or had no effect, respectively. The AS 19 facilitatory effect was reversed by SB-269970, but not by the selective 5-HT1A antagonist WAY100635. Amnesia induced by scopolamine (cholinergic antagonist) or dizocilpine (NMDA antagonist) was also reversed by AS 19. Certainly, reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool. Having mentioned that, it should be noticed that together these data are providing further support to the notion of the 5-HT7 receptors role in memory formation. Importantly, this 5-HT7 receptor agonist AS 19 appears to represent a step forward respect to the notion that potent and selective 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer's disease.

Partial agonists for α4β2 nicotinic receptors stimulate dopaminergic neuron firing with relatively enhanced maximal effects

PubMed Central

Chen, Ying; Broad, Lisa M; Phillips, Keith G; Zwart, Ruud

2012-01-01

BACKGROUND AND PURPOSE Partial agonists selective for α4β2 nicotinic ACh receptors have been developed for smoking cessation as they induce weak activation of native α4β2* receptors and inhibit effect of nicotine. However, it is unclear whether at brain functions there is an existence of receptor reserve that allows weak receptor activation to induce maximum physiological effects. We assessed the extent of α4β2 partial agonist-induced increase of firing rate in dopaminergic neurons and evaluated the influence of receptor reserve. EXPERIMENTAL APPROACH The relative maximal effects and potencies of six nicotinic agonists were assessed on recombinant human α4β2 and α7 receptors expressed in mammalian cell lines by measuring calcium influx. Agonist-induced increase of the spontaneous firing rate of dopaminergic neurons was recorded using microelectrodes in the ventral tegmental area of rat brain slices. KEY RESULTS All α4β2 partial and full agonists increased the firing rate concentration-dependently. Their sensitivity to subtype-selective antagonists showed predominant activation of native α4β2* receptors. However, partial agonists with relative maximal effects as low as 33% on α4β2 receptors maximally increased the firing rate and induced additional depolarization block of firing, demonstrating that partial activation of receptors caused the maximum increase in firing rate in the presence of a receptor reserve. CONCLUSIONS AND IMPLICATIONS Partial α4β2 agonists induced relatively enhanced effects on the firing rate of dopaminergic neurons, and the effect was mainly attributed to the existence of native α4β2* receptor reserve. The results have implications in the understanding of physiological effects and therapeutic efficacies of α4β2 partial agonists. PMID:21838750

Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

PubMed Central

Guo, Dong; Mulder-Krieger, Thea; IJzerman, Adriaan P; Heitman, Laura H

2012-01-01

BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. PMID:22324512

N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisone, G.; Berthold, M.; Bedecs, K.

1989-12-01

The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis.more » Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.« less

The G‐protein biased partial κ opioid receptor agonist 6′‐GNTI blocks hippocampal paroxysmal discharges without inducing aversion

PubMed Central

Zangrandi, Luca; Burtscher, Johannes; MacKay, James P; Colmers, William F

2016-01-01

Background and Purpose With a prevalence of 1–2%, epilepsies belong to the most frequent neurological diseases worldwide. Although antiepileptic drugs are available since several decades, the incidence of patients that are refractory to medication is still over 30%. Antiepileptic effects of κ opioid receptor (κ receptor) agonists have been proposed since the 1980s. However, their clinical use was hampered by dysphoric side effects. Recently, G‐protein biased κ receptor agonists were developed, suggesting reduced aversive effects. Experimental Approach We investigated the effects of the κ receptor agonist U‐50488H and the G‐protein biased partial κ receptor agonist 6′‐GNTI in models of acute seizures and drug‐resistant temporal lobe epilepsy and in the conditioned place avoidance (CPA) test. Moreover, we performed slice electrophysiology to understand the functional mechanisms of 6′‐GNTI. Key Results As previously shown for U‐50488H, 6′‐GNTI markedly increased the threshold for pentylenetetrazole‐induced seizures. All treated mice displayed reduced paroxysmal activity in response to U‐50488H (20 mg·kg−1) or 6′‐GNTI (10–30 nmoles) treatment in the mouse model of intra‐hippocampal injection of kainic acid. Single cell recordings on hippocampal pyramidal cells revealed enhanced inhibitory signalling as potential mechanisms causing the reduction of paroxysmal activity. Effects of 6′‐GNTI were blocked in both seizure models by the κ receptor antagonist 5′‐GNTI. Moreover, 6′‐GNTI did not induce CPA, a measure of aversive effects, while U‐50488H did. Conclusions and Implications Our data provide the proof of principle that anticonvulsant/antiseizure and aversive effects of κ receptor activation can be pharmacologically separated in vivo. PMID:26928671

The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

NASA Astrophysics Data System (ADS)

Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

1987-07-01

Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, Martyn C; Boyfield, Izzy; Brown, Tony; Hagan, Jim J; Middlemiss, Derek N

1999-01-01

The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF-104557 (4-[2-(propylamino)ethyl]-2-(3H) indolone), SKF-97930 (4-carboxy-2-(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry.All the receptor agonists tested (ropinirole, SKF-104557, SKF-97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members.At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF-104557, were partial agonists at the hD4 receptor.Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s.The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities.The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF-104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound. PMID:10455328

Agonist-Directed Desensitization of the β2-Adrenergic Receptor

PubMed Central

Goral, Vasiliy; Jin, Yan; Sun, Haiyan; Ferrie, Ann M.; Wu, Qi; Fang, Ye

2011-01-01

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level. PMID:21541288

L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

PubMed Central

Hargreaves, R. J.; McKnight, A. T.; Scholey, K.; Newberry, N. R.; Street, L. J.; Hutson, P. H.; Semark, J. E.; Harley, E. A.; Patel, S.; Freedman, S. B.

1992-01-01

1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1422595

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

PubMed

Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

2004-03-26

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

General, kappa, delta and mu opioid receptor antagonists mediate feeding elicited by the GABA-B agonist baclofen in the ventral tegmental area and nucleus accumbens shell in rats: reciprocal and regional interactions.

PubMed

Miner, Patricia; Shimonova, Lyudmila; Khaimov, Arthur; Borukhova, Yaffa; Ilyayeva, Ester; Ranaldi, Robert; Bodnar, Richard J

2012-03-14

Food intake is significantly increased following administration of agonists of GABA and opioid receptors into the nucleus accumbens shell (NACs) and ventral tegmental area (VTA). GABA-A or GABA-B receptor antagonist pretreatment within the VTA or NACs differentially affects mu-opioid agonist-induced feeding elicited from the same site. Correspondingly, general or selective opioid receptor antagonist pretreatment within the VTA or NACs differentially affects GABA agonist-induced feeding elicited from the same site. Regional interactions have been evaluated in feeding studies by administering antagonists in one site prior to agonist administration in a second site. Thus, opioid antagonist-opioid agonist and GABA antagonist-GABA agonist feeding interactions have been identified between the VTA and NACs. However, pretreatment with GABA-A or GABA-B receptor antagonists in the VTA failed to affect mu opioid agonist-induced feeding elicited from the NACs, and correspondingly, these antagonists administered in the NACs failed to affect mu opioid-induced feeding elicited from the VTA. To evaluate whether regional and reciprocal VTA and NACs feeding interactions occur for opioid receptor modulation of GABA agonist-mediated feeding, the present study examined whether feeding elicited by the GABA-B agonist, baclofen microinjected into the NACs was dose-dependently blocked by pretreatment with general (naltrexone: NTX), mu (beta-funaltrexamine: BFNA), kappa (nor-binaltorphamine: NBNI) or delta (naltrindole: NTI) opioid antagonists in the VTA, and correspondingly, whether VTA baclofen-induced feeding was dose-dependently blocked by NACs pretreatment with NTX, BFNA, NBNI or NTI in rats. Bilateral pairs of cannulae aimed at the VTA and NACs were stereotaxically implanted in rats, and their food intakes were assessed following vehicle and baclofen (200 ng) in each site. Baclofen produced similar magnitudes of increased food intake following VTA and NACs treatment. Baclofen administration in the VTA or NACs was also preceded by administration of NTX (0.1, 1, 5 μg, 0.5 h), BFNA (0.4, 4 μg, 24 h), NBNI (0.6, 6 μg, 0.5 h) or NTI (0.4, 4 μg, 0.5 h) into the other site with intake measured 1, 2 and 4 h after agonist treatment. VTA NTX significantly reduced NACs baclofen-induced feeding. Correspondingly, NACs NTX significantly reduced VTA baclofen-induced feeding, indicating a robust and bidirectional general opioid and GABA-B receptor feeding interaction. Whereas the high, but not low VTA BFNA dose reduced NACs baclofen-induced feeding, NACs BFNA failed to affect VTA baclofen-induced feeding, indicating a unidirectional mu opioid and GABA-B receptor feeding interaction. Whereas VTA NBNI at both doses reduced NACs baclofen-induced feeding, the high, but not low NACs NBNI dose significantly reduced VTA baclofen-induced feeding, indicating a bidirectional kappa opioid and GABA-B receptor feeding interaction. Whereas VTA NTI only transiently reduced NACs baclofen-induced feeding, NACs NTI failed to affect VTA baclofen-induced feeding, indicating a weak unidirectional delta opioid and GABA-B receptor interaction. Whereas administration of NTX or BFNA into the NACs or VTA marginally reduced spontaneous food intake, NBNI or NTI into the same sites failed to alter food intake alone. Therefore, the present study suggests that GABA employs a distributed brain network in mediating its ingestive effects that is dependent upon intact opioid receptor signaling with kappa opioid receptors more involved than mu and delta opioid receptors underlying these regional effects. An alternative hypothesis to be considered is that these effects could be the sum of two independent drug effects (opioid antagonists decreasing and baclofen increasing food intake). Copyright © 2012 Elsevier B.V. All rights reserved.

Structure-activity relationships of the unique and potent agouti-related protein (AGRP)-melanocortin chimeric Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template.

PubMed

Wilczynski, Andrzej; Wilson, Krista R; Scott, Joseph W; Edison, Arthur S; Haskell-Luevano, Carrie

2005-04-21

The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.

Anti-epileptic activity of group II metabotropic glutamate receptor agonists (--)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (--)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795).

PubMed

Moldrich, R X; Jeffrey, M; Talebi, A; Beart, P M; Chapman, A G; Meldrum, B S

2001-07-01

The selective group II metabotropic glutamate receptor (mGlu(2/3)) agonists (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795) have been evaluated as anti-epileptic drugs in dilute brown agouti (DBA/2) mice, lethargic (lh/lh) mice, genetically epilepsy-prone-9 (GEP) rats and amygdala-kindled rats. Sound-induced clonic seizures in DBA/2 mice were transiently inhibited by both agonists intracerebroventricularly (i.c.v.), LY379268 ED(50)=0.08 [0.02-0.33]nmol and LY389795 ED(50)=0.82 [0.27-3.24]nmol or intraperitoneally (i.p.), LY379268 ED(50)=2.9 [0.9-9.6]mg/kg and LY389795 ED(50)=3.4 [1.0-11.7]mg/kg. Both mGlu(2/3) agonists inhibited seizures induced by the group I mGlu receptor agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG), where LY379268, i.c.v. ED(50)=0.3 [0.02-5.0]pmol and LY389795, i.c.v. ED(50)=0.03 [0.05-0.19]nmol. The spike and wave discharge (SWD) duration of absence seizures in lh/lh mice was significantly reduced by both agonists at 1 and 10nmol (i.c.v.) up to 90min following infusion. The electrically induced seizure score and afterdischarge duration of amygdala-kindled rats was partially inhibited by the agonists 30min after i.p. injection of 10mg/kg. The agonists did not inhibit sound-induced seizures in GEP rats (0.1-1mg/kg, 30min 1h, i.p.), but were proconvulsant following sound stimulus (> or =0.1mg/kg). These findings identify a potential role for mGlu(2/3) agonists in the amelioration of generalised and partial epileptic seizures.

In vivo neurochemical evidence that delta1-, delta2- and mu2-opioid receptors, but not mu1-opioid receptors, inhibit acetylcholine efflux in the nucleus accumbens of freely moving rats.

PubMed

Kiguchi, Yuri; Aono, Yuri; Watanabe, Yuriko; Yamamoto-Nemoto, Seiko; Shimizu, Kunihiko; Shimizu, Takehiko; Kosuge, Yasuhiro; Waddington, John L; Ishige, Kumiko; Ito, Yoshihisa; Saigusa, Tadashi

2016-10-15

Cholinergic neurons in the nucleus accumbens express delta- and mu-opioid receptors that are thought to inhibit neural activity. Delta- and mu-opioid receptors are divided into delta1- and delta2-opioid receptors and mu1- and mu2-opioid receptors, respectively. We analysed the roles of delta- and mu-opioid receptor subtypes in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. Other than naloxonazine, given intraperitoneally, delta- and mu-opioid receptor ligands were administered intracerebrally through the dialysis probe. Doses of these compounds indicate total amount (mol) over an infusion time of 30-60min. To monitor basal acetylcholine, a low concentration of physostigmine (50nM) was added to the perfusate. The delta1-opioid receptor agonist DPDPE (3 and 300pmol) and delta2-opioid receptor agonist deltorphin II (3 and 30pmol) decreased accumbal acetylcholine in a dose-related manner. DPDPE (300pmol)- and deltorphin II (3pmol)-induced reductions in acetylcholine were each inhibited by the delta1-opioid receptor antagonist BNTX (0.3pmol) and delta2-opioid receptor antagonist naltriben (15pmol), respectively. The mu-opioid receptor agonists endomorphin-1 and endomorphin-2 (6 and 30nmol) decreased acetylcholine in a dose-related manner. Endomorphin-1- and endomorphin-2 (30nmol)-induced reductions in acetylcholine were prevented by the mu-opioid receptor antagonist CTOP (3nmol). The mu1-opioid receptor antagonist naloxonazine (15mg/kg ip), which inhibits endomorphin-1 (15nmol)-induced accumbal dopamine efflux, did not alter endomorphin-1- or endomorphin-2 (30nmol)-induced reductions in acetylcholine efflux. This study provides in vivo evidence for delta1-, delta2- and mu2-opioid receptors, but not mu1-opioid receptors, that inhibit accumbal cholinergic neural activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of EMD 128 130 occupancy of the 5-HT1A and the D2 receptor: a human PET study with [11C]WAY-100635 and [11C]raclopride.

PubMed

Rabiner, Eugenii A; Gunn, Roger N; Wilkins, Martin R; Sedman, Ewen; Grasby, Paul M

2002-09-01

The use of so-called, atypical antipsychotic medication is becoming more widespread in the treatment of psychotic disorders. EMD 128 130 is a novel compound acting as an agonist at the 5-HT1A receptor, and as an antagonist at the dopamine-2 (D2) receptor. This dual action may confer additional benefits over selective D2 antagonists in the treatment of psychotic disorders. In this study, we investigated the occupancy of EMD 128 130 in vivo at the human D2 and 5-HT1A receptors with positron emission tomography using the radiotracers [11C]raclopride and [11C]WAY-100635. Seven healthy volunteers were examined before and after 5 days of treatment with EMD 128 130, administered in an incremental dose building up to 50 mg, b.d. A significant occupancy was demonstrated at the human D2 receptor (40% following a dose of 50 mg, b.d.) while there was no consistent effect observed at the 5-HT1A receptor, despite a similar affinity of EMD 128 130 for cloned human D2 and 5-HT1A receptors, and the presence of typical, central 5-HT1A agonist side-effects. The differential effects of EMD 128 130 at the D2 and the 5-HT1A receptor (antagonist at D2 receptor, agonist at the 5-HTIA receptor) may explain the differences in occupancy observed.

Neurokinin receptors in the rabbit iris sphincter characterised by novel agonist ligands.

PubMed

Hall, J M; Mitchell, D; Morton, I K

1991-06-18

We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist delta-amino valeryl-[L-Pro9,N-Me Leu10]SP-(7-11) (GR73632) and the NK-3 receptor-selective agonist succ-[Asp6,N-Me-Phe8] SP-(6-11) (senktide) were both very active (concentration range 0.032 pM-10 nM and 0.1 pM-32 nM respectively), and were 933 and 16.6 times more potent than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys3,Gly8-R-gamma-lactam,Leu9]NKA-(3-10) (GR64349) was active only at the highest concentrations tested (3.2 nM-32 microM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GR64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study.

The selective V1a receptor agonist selepressin (FE 202158) blocks vascular leak in ovine severe sepsis

PubMed Central

Wiśniewska, Halina; Traber, Lillian D.; Lin, ChiiDean; Fan, Juanjuan; Hawkins, Hal K.; Cox, Robert A.; Wiśniewski, Kazimierz; Schteingart, Claudio D.; Landry, Donald W.; Rivière, Pierre J.-M.; Traber, Daniel L.

2014-01-01

Objective To determine if the selective vasopressin type 1a receptor (V1aR) agonist selepressin (FE 202158) is as effective as the mixed V1a/V2 receptor (V1aR/V2R) agonist vasopressor hormone arginine vasopressin (AVP) when used as a titrated first-line vasopressor therapy in an ovine model of Pseudomonas aeruginosa pneumonia-induced severe sepsis. Design Prospective, randomized, controlled laboratory experiment. Setting University animal research facility. Subjects Forty-five chronically instrumented sheep. Interventions Sheep were anesthetized, insufflated with cooled cotton smoke via tracheostomy, and P. aeruginosa were instilled into their airways. They were then placed on assisted ventilation, awakened, and resuscitated with lactated Ringer's solution titrated to maintain hematocrit ± 3% from baseline levels. If, despite fluid management, mean arterial pressure (MAP) fell by > 10 mm Hg from baseline levels, a continuous i.v. infusion of AVP or selepressin was titrated to raise and maintain MAP within 10 mm Hg of baseline. Effects of combination treatment of selepressin with the selective V2R agonist desmopressin were similarly investigated. Measurements and Main Results In septic sheep, MAP fell by ~30 mm Hg, systemic vascular resistance index (SVRI) decreased by ~50%, and ~7 L of fluid were retained over 24 h; this fluid accumulation was partially reduced by AVP and almost completely blocked by selepressin; combined infusion of selepressin and desmopressin increased fluid accumulation to levels similar to AVP treatment. Conclusions Resuscitation with the selective V1aR agonist selepressin blocked vascular leak more effectively than the mixed V1aR/V2R agonist AVP because of its lack of agonist activity at the V2R. PMID:24674922

Dopamine D1 and D3 Receptors Modulate Heroin-Induced Cognitive Impairment through Opponent Actions in Mice

PubMed Central

Zhu, Yongsheng; Wang, Yunpeng; Wei, Shuguang; Zhang, Hongbo; Yan, Peng; Li, Yunxiao; Qiao, Xiaomeng; Yin, Fangyuan

2017-01-01

Abstract Background: Chronic abuse of heroin leads to long-lasting and complicated cognitive impairment. Dopamine receptors are critically involved in the impulsive drug-driven behavior and the altered attention, processing speed, and mental flexibility that are associated with higher relapse rates. However, the effects of the different dopamine receptors and their possible involvement in heroin-induced cognitive impairment remain unclear. Methods: The 5-choice serial reaction time task was used to investigate the profiles of heroin-induced cognitive impairment in mice. The expression levels of dopamine D1- and D2-like receptors in the prefrontal cortex, nucleus accumbens, and caudate-putamen were determined. The effects of dopamine receptors on heroin-induced impulsivity in the 5-choice serial reaction time task were examined by agonist/antagonist treatment on D1 or D3 receptor mutant mice. Results: Systemic heroin administration influences several variables in the 5-choice serial reaction time task, most notably premature responses, a measure of motor impulsivity. These behavioral impairments are associated with increased D1 receptor and decreased D3 receptor mRNA and protein levels in 3 observed brain areas. The heroin-evoked increase in premature responses is mimicked by a D1 agonist and prevented by a D1 antagonist or genetic ablation of the D1 receptor gene. In contrast, a D3 agonist decreases both basal and heroin-evoked premature responses, while genetic ablation of the D3 receptor gene results in increased basal and heroin-evoked premature responses. Conclusions: Heroin-induced impulsive behavior in the 5-choice serial reaction time task is oppositely modulated by D1 and D3 receptor activation. The D1 receptors in the cortical-mesolimbic region play an indispensable role in modulating such behaviors. PMID:27815417

Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

PubMed

Harland, Aubrie A; Yeomans, Larisa; Griggs, Nicholas W; Anand, Jessica P; Pogozheva, Irina D; Jutkiewicz, Emily M; Traynor, John R; Mosberg, Henry I

2015-11-25

In a previously described peptidomimetic series, we reported the development of bifunctional μ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.

An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

PubMed Central

Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

2016-01-01

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297

Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Yamada, M; Yamada, M; Lombet, A; Forgez, P; Rostène, W

1998-01-01

Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.

Electrophysiological and autoradiographical evidence of V1 vasopressin receptors in the lateral septum of the rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raggenbass, M.; Tribollet, E.; Dreifuss, J.J.

1987-11-01

Extracellular recordings were obtained from single neurons located in the lateral septum, an area known to receive a vasopressinergic innervation in the rat brain. Approximately half of the neurons tested responded to 8-L-arginine vasopressin (AVP) by a marked increase in firing rate at concentrations greater than 1 nM. The effect of vasopressin was blocked by synthetic structural analogues possessing antagonistic properties on peripheral vasopressin and oxytocin receptors. Oxytocin was much less potent than vasopressin in firing septal neurons, and a selective oxytocic agonist was totally ineffective. The action of vasopressin on neuronal firing was mimicked by the vasopressor agonist (2-phenylalanine,8-ornithine)vasotocinmore » but not by the selective antidiuretic agonist 1-deamino(8-D-arginine)vasopressin. In a parallel study, sites that bind (/sup 3/H)AVP at low concentration (1.5 nM) were found by in vitro autoradiography in the lateral septum. Adjacent sections were also incubated with 1.5 mM (/sup 3/H)AVP and, in addition, with 100 nM (2-phenylalanine,8-ornithine)vasotocin or 1-deamino(8-D-arginine)vasopressin--i.e., the same compounds as those used for the electrophysiological study. Results showed that the vasopressor agonist, but not the antidiuretic agonist, displaced (/sup 3/H)AVP, thus indicating that the vasopressin binding sites detected by autoradiography in the septum were V1 (vasopressor type) rather than V2 (antidiuretic type) receptors. Based on the electrophysiological evidence, we conclude that these receptors, when occupied, lead to increased firing of lateral septal neurons.« less

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae)

PubMed Central

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Alecjev; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K.; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-01-01

Ethnopharmacological relevance Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q’eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. Aim of the study To investigate the estrogenic and serotonergic activities of P. hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. Materials and methods The effects of the P. hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Results Three butenolides including one new compound (1) were isolated from the leaves of P. hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT7 with IC50 values of 16.1 and 8.3 μM, respectively, and using GTP shift assays, compound 1 was found to be a partial agonist of the 5-HT7 receptor. The P. hispidum leaf extracts, as well as compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Conclusions Extracts of P. hispidum act as agonists of the ER and 5-HT7 receptors. Compound 1, a new natural product, identified as 9, 10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT7 agonist. Compounds 2 and 3 are reported for the first time in P. hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 μM. These activities are consistent with the Q’eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. PMID:20304039

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae).

PubMed

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Aleksej; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-05-27

Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q'eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. To investigate the estrogenic and serotonergic activities of Piper hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. The effects of the Piper hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Three butenolides including one new compound (1) were isolated from the leaves of Piper hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT(7) with IC(50) values of 16.1 and 8.3 microM, respectively, and using GTP shift assays, Compound 1 was found to be a partial agonist of the 5-HT(7) receptor. The Piper hispidum leaf extracts, as well as Compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Extracts of Piper hispidum act as agonists of the ER and 5-HT(7) receptors. Compound 1, a new natural product, identified as 9,10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT(7) agonist. Compounds 2 and 3 are reported for the first time in Piper hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 microM. These activities are consistent with the Q'eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28.

PubMed

Hulshof, Janneke W; Casarosa, Paola; Menge, Wiro M P B; Kuusisto, Leena M S; van der Goot, Henk; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

2005-10-06

US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.

Presynaptic selectivity of a ligand for serotonin 1A receptors revealed by in vivo PET assays of rat brain.

PubMed

Saijo, Takeaki; Maeda, Jun; Okauchi, Takashi; Maeda, Jun-ichi; Morio, Yasunori; Kuwahara, Yasuhiro; Suzuki, Masayuki; Goto, Nobuharu; Fukumura, Toshimitsu; Suhara, Tetsuya; Higuchi, Makoto

2012-01-01

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT(1A)) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT(1A) receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT(1A) receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT(1A) receptors. In addition, [(35)S]guanosine 5'-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT(1A) receptors. This finding has lent support to reports that diverse partial agonists for 5-HT(1A) receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.

Presynaptic Selectivity of a Ligand for Serotonin 1A Receptors Revealed by In Vivo PET Assays of Rat Brain

PubMed Central

Okauchi, Takashi; Maeda, Jun-ichi; Morio, Yasunori; Kuwahara, Yasuhiro; Suzuki, Masayuki; Goto, Nobuharu; Fukumura, Toshimitsu; Suhara, Tetsuya; Higuchi, Makoto

2012-01-01

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT1A) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT1A receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT1A receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT1A receptors. In addition, [35S]guanosine 5′-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT1A receptors. This finding has lent support to reports that diverse partial agonists for 5-HT1A receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants. PMID:22880045

The group II metabotropic glutamate receptor agonist LY354740 and the D2 receptor antagonist haloperidol reduce locomotor hyperactivity but fail to rescue spatial working memory in GluA1 knockout mice.

PubMed

Boerner, Thomas; Bygrave, Alexei M; Chen, Jingkai; Fernando, Anushka; Jackson, Stephanie; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H; Harrison, Paul J; Gilmour, Gary; Bannerman, David M; Sanderson, David J

2017-04-01

Group II metabotropic glutamate receptor agonists have been suggested as potential anti-psychotics, at least in part, based on the observation that the agonist LY354740 appeared to rescue the cognitive deficits caused by non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonists, including spatial working memory deficits in rodents. Here, we tested the ability of LY354740 to rescue spatial working memory performance in mice that lack the GluA1 subunit of the AMPA glutamate receptor, encoded by Gria1, a gene recently implicated in schizophrenia by genome-wide association studies. We found that LY354740 failed to rescue the spatial working memory deficit in Gria1 -/- mice during rewarded alternation performance in the T-maze. In contrast, LY354740 did reduce the locomotor hyperactivity in these animals to a level that was similar to controls. A similar pattern was found with the dopamine receptor antagonist haloperidol, with no amelioration of the spatial working memory deficit in Gria1 -/- mice, even though the same dose of haloperidol reduced their locomotor hyperactivity. These results with LY354740 contrast with the rescue of spatial working memory in models of glutamatergic hypofunction using non-competitive NMDAR antagonists. Future studies should determine whether group II mGluR agonists can rescue spatial working memory deficits with other NMDAR manipulations, including genetic models and other pharmacological manipulations of NMDAR function. © 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists

NASA Astrophysics Data System (ADS)

de Marino, Simona; Carino, Adriana; Masullo, Dario; Finamore, Claudia; Marchianò, Silvia; Cipriani, Sabrina; di Leva, Francesco Saverio; Catalanotti, Bruno; Novellino, Ettore; Limongelli, Vittorio; Fiorucci, Stefano; Zampella, Angela

2017-02-01

Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

Sigma-1 receptor chaperone and brain-derived neurotrophic factor: emerging links between cardiovascular disease and depression.

PubMed

Hashimoto, Kenji

2013-01-01

Epidemiological studies have demonstrated a close relationship between depression and cardiovascular disease (CVD). Although it is known that the central nervous system (CNS) contributes to this relationship, the detailed mechanisms involved in this process remain unclear. Recent studies suggest that the endoplasmic reticulum (ER) molecular chaperone sigma-1 receptor and brain-derived neurotrophic factor (BDNF) play a role in the pathophysiology of CVD and depression. Several meta-analysis studies have showed that levels of BDNF in the blood of patients with major depressive disorder (MDD) are lower than normal controls, indicating that blood BDNF might be a biomarker for depression. Furthermore, blood levels of BDNF in patients with CVD are also lower than normal controls. A recent study using conditional BDNF knock-out mice in animal models of myocardial infarction highlighted the role of CNS-mediated mechanisms in the cardioprotective effects of BDNF. In addition, a recent study shows that decreased levels of sigma-1 receptor in the mouse brain contribute to the association between heart failure and depression. Moreover, sigma-1 receptor agonists, including the endogenous neurosteroid dehydroepiandosterone (DHEA) and the selective serotonin reuptake inhibitor (SSRI) fluvoxamine, show potent cardioprotective and antidepressive effects in rodents, via sigma-1 receptor stimulation. Interestingly, agonist activation of sigma-1 receptors increased the secretion of mature BDNF from its precursor proBDNF via chaperone activity in the ER. Given the role of ER stress in the pathophysiology of CVD and MDD, the author will discuss the potential link between sigma-1 receptors and BDNF-TrkB pathway in the pathophysiology of these two diseases. Finally, the author will make a case for potent sigma-1 receptor agonists and TrkB agonists as new potential therapeutic drugs for depressive patients with CVD. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis and pharmacological characterization of novel inverse agonists acting on the viral-encoded chemokine receptor US28.

PubMed

Hulshof, Janneke W; Vischer, Henry F; Verheij, Mark H P; Fratantoni, Silvina A; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

2006-11-01

G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

Actions of novel antipsychotic agents on apomorphine-induced PPI disruption: influence of combined serotonin 5-HT1A receptor activation and dopamine D2 receptor blockade.

PubMed

Auclair, Agnès L; Kleven, Mark S; Besnard, Joël; Depoortère, Ronan; Newman-Tancredi, Adrian

2006-09-01

The dopamine D1/D2 agonist apomorphine (0.63 mg/kg) disrupted prepulse inhibition (PPI) of acoustic startle in rats, a model of sensorimotor gating deficits observed in schizophrenia. All current antipsychotics, which antagonize D2 receptors, prevent this apomorphine-induced deficit. A novel class of antipsychotics possesses, in addition to D2 antagonist property, various levels of 5-HT1A agonist activity. Considering that the latter itself produces PPI deficits, it appeared necessary to assess the potential of this novel class of antipsychotics to reverse apomorphine-PPI deficits. Potent D2 antagonists, like haloperidol (0.63-2.5 mg/kg), risperidone (0.63-10 mg/kg), and olanzapine (0.63-40 mg/kg) prevented apomorphine PPI disruption. The atypical antipsychotics, clozapine (40 mg/kg), nemonapride (0.01-2.5 mg/kg), ziprasidone (10 mg/kg), and aripiprazole (0.01 and 10 mg/kg), which all exhibit 5-HT1A agonist properties, reversed PPI deficits at some doses only, whereas the anti-dyskinetic agent sarizotan (0.16-10 mg/kg), an efficacious 5-HT1A agonist, did not. New generation antipsychotics with marked 5-HT1A agonist properties, such as SLV313 and SSR181507 (0.0025-10 mg/kg and 0.16-10 mg/kg, respectively) did not reverse these deficits whereas bifeprunox (0.04-2.5 mg/kg) did. To reveal the contribution of 5-HT1A agonist properties in the lack of effects of SLV313 and SSR181507, we pretreated rats with the 5-HT1A antagonist WAY100635 (0.63 mg/kg). Under these conditions, significant reversal of PPI deficit was observed, indicating that D2 antagonist properties of SLV313 and SSR181507 are now sufficient to overcome the disruptive effects of apomorphine. To summarize, antipsychotics possessing agonist efficacy at 5-HT1A receptors exhibit diverse profiles against apomorphine-induced PPI deficits, depending on the balance between D2 and 5-HT1A activities, suggesting that they may display distinct activity on some aspects of gating deficits in schizophrenic patients.

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

The effects of the synthetic cannabinoid receptor agonists, WIN55,212-2 and CP55,940, on salicylate-induced tinnitus in rats.

PubMed

Zheng, Yiwen; Stiles, Lucy; Hamilton, Emma; Smith, Paul F; Darlington, Cynthia L

2010-09-01

Previous studies in animals and humans have shown that, in some cases at least, anti-epileptic drugs can reduce the severity of tinnitus. Given that cannabinoid receptor agonists have been shown to exert anti-epileptic effects in some circumstances, we investigated whether two synthetic CB(1)/CB(2) receptor agonists, WIN55,212-2, and CP55,940, could inhibit the behavioural manifestations of salicylate-induced tinnitus in rats in a conditioned suppression task. We found that neither WIN55,212-2 (3.0 mg/kg s.c) nor CP55,940 (0.1 or 0.3 mg/kg s.c), significantly reduced conditioned behaviour associated with tinnitus. However, both 3 mg/kg WIN55,212-2 and 0.3 mg/kg CP55,940 did significantly increase tinnitus-related behaviour compared to the vehicle control groups. These results suggest that cannabinoid receptor agonists may not be useful in the treatment of salicylate-induced tinnitus and that at certain doses, they could actually exacerbate the condition. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis.

PubMed

Jenkinson, K M; Southwell, B R; Furness, J B

1999-01-01

1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor.

Differential effects of subtype-specific nicotinic acetylcholine receptor agonists on early and late hippocampal LTP.

PubMed

Kroker, Katja S; Rast, Georg; Rosenbrock, Holger

2011-12-05

Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer's and Parkinson's diseases, Tourette's syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer's disease patients. These are α4β2 and α7 nicotinic acetylcholine receptor agonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptor agonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptor agonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptor agonists. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro pharmacology of aripiprazole, its metabolite and experimental dopamine partial agonists at human dopamine D2 and D3 receptors.

PubMed

Tadori, Yoshihiro; Forbes, Robert A; McQuade, Robert D; Kikuchi, Tetsuro

2011-10-15

Aripiprazole is the first dopamine D(2)/D(3) receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D(2) and serotonin 5-HT(1A) receptor partial agonist activities have been postulated to confer clinical efficacy without marked sedation, and a relatively favorable overall side-effect profile. Using aripiprazole's unique profile as a benchmark for new dopamine partial agonist development may facilitate discovery of new antipsychotics. We conducted an in vitro comparative analysis between aripiprazole, and its human metabolite OPC-14857 (7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl)butoxy)-2(1H)-quinolinone)); RGH-188 (trans-1-[4-[2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl]cyclohexyl]-3,3-dimethylurea), and its metabolite didesmethyl-RGH-188 (DDM-RGH-188); as well as bifeprunox, sarizotan, N-desmethylclozapine (NDMC; clozapine metabolite), and SDZ 208-912 (N-[(8α)-2-chloro-6-methylergolin-8-yl]-2,2-dimethylpropanamide). In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cell lines expressing D(2S), D(2L), D(3) Ser-9 and D(3) Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D(2)/D(3) receptor partial agonists. Aripiprazole's intrinsic activity at dopamine D(2S) and D(2L) receptors was similar to that of OPC-14857 and RGH-188; lower than that of dopamine and bifeprunox; and higher than that of DDM-RGH-188, SDZ 208-912, sarizotan, and NDMC. Aripiprazole's intrinsic activity at dopamine D(3) Ser-9 and D(3) Gly-9 receptors was similar to that of OPC-14857 and sarizotan; lower than that of dopamine, bifeprunox, RGH-188 and DDM-RGH-188; and higher than that of SDZ 208-912 and NDMC. A consolidated assessment of these findings may help defining the most appropriate magnitude of intrinsic activity at dopamine D(2)/D(3) receptors for clinical efficacy and safety. Copyright © 2011 Elsevier B.V. All rights reserved.

A structure-function study of PACAP using conformationally-restricted analogs: identification of PAC1 receptor-selective PACAP agonists

PubMed Central

Ramos-Álvarez, Irene; Mantey, Samuel A.; Nakamura, Taichi; Nuche-Berenguer, Bernardo; Moreno, Paola; Moody, Terry W.; Maderdrut, Jerome L.; Coy, David H.; Jensen, Robert T.

2015-01-01

Pituitary adenylate-cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure-function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally-restricted PACAP -analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22 ,28,34,38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to-103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases. PMID:25698233

Design, synthesis, and in vivo SAR of a novel series of pyrazolines as potent selective androgen receptor modulators.

PubMed

Zhang, Xuqing; Li, Xiaojie; Allan, George F; Sbriscia, Tifanie; Linton, Olivia; Lundeen, Scott G; Sui, Zhihua

2007-08-09

A novel series of pyrazolines 2 have been designed, synthesized, and evaluated by in vivo screening as tissue-selective androgen receptor modulators (SARMs). Structure-activity relationships (SAR) were investigated at the R1 to R6 positions as well as the core pyrazoline ring and the anilide linker. Overall, strong electron-withdrawing groups at the R1 and R2 positions and a small group at the R5 and R6 position are optimal for AR agonist activity. The (S)-isomer of 7c exhibits more potent AR agonist activity than the corresponding (R)-isomer. (S)-7c exhibited an overall partial androgenic effect but full anabolic effect via oral administration in castrated rats. It demonstrated a noticeable antiandrogenic effect on prostate in intact rats with endogenous testosterone. Thus, (S)-7c is a tissue-selective nonsteroidal androgen receptor modulator with agonist activity on muscle and mixed agonist and antagonist activity on prostate.

Renoprotective Effects of AVE0991, a Nonpeptide Mas Receptor Agonist, in Experimental Acute Renal Injury

PubMed Central

Barroso, Lívia Corrêa; Silveira, Kátia Daniela; Lima, Cristiano Xavier; Borges, Valdinéria; Bader, Michael; Rachid, Milene; Santos, Robson Augusto Souza; Souza, Danielle Gloria; Simões e Silva, Ana Cristina; Teixeira, Mauro Martins

2012-01-01

Renal ischemia and reperfusion (I/R) is the major cause of acute kidney injury in hospitalized patients. Mechanisms underlying reperfusion-associated injury include recruitment and activation of leukocytes and release of inflammatory mediators. In this study, we investigated the renal effects of acute administration of AVE0991, an agonist of Mas, the angiotensin-(1–7) receptor, the angiotensin-(1–7) receptor, in a murine model of renal I/R. Male C57BL/6 wild-type or Mas−/− mice were subjected to 30 min of bilateral ischemia and 24 h of reperfusion. Administration of AVE0991 promoted renoprotective effects, as seen by improvement of function, decreased tissue injury, prevention of local and remote leucocyte infiltration, and release of the chemokine, CXCL1. I/R injury was similar in WT and Mas−/− mice, suggesting that endogenous activation of this receptor does not control renal damage under baseline conditions. In conclusion, pharmacological interventions using Mas receptor agonists may represent a therapeutic opportunity for the treatment of renal I/R injury. PMID:22319645

The CB2 cannabinoid agonist AM-1241 prolongs survival in a transgenic mouse model of amyotrophic lateral sclerosis when initiated at symptom onset

PubMed Central

Shoemaker, Jennifer L.; Seely, Kathryn A.; Reed, Ronald L.; Crow, John P.; Prather, Paul L.

2010-01-01

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, paralysis and death within 2–5 years of diagnosis. Currently, no effective pharmacological agents exist for the treatment of this devastating disease. Neuroinflammation may accelerate the progression of ALS. Cannabinoids produce anti-inflammatory actions via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), and delay the progression of neuroinflammatory diseases. Additionally, CB2 receptors, which normally exist primarily in the periphery, are dramatically up-regulated in inflamed neural tissues associated with CNS disorders. In G93A-SOD1 mutant mice, the most well-characterized animal model of ALS, endogenous cannabinoids are elevated in spinal cords of symptomatic mice. Furthermore, treatment with non-selective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. We demonstrate that mRNA, receptor binding and function of CB2, but not CB1, receptors are dramatically and selectively up-regulated in spinal cords of G93A-SOD1 mice in a temporal pattern paralleling disease progression. More importantly, daily injections of the selective CB2 agonist AM-1241, initiated at symptom onset, increase the survival interval after disease onset by 56%. Therefore, CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS. PMID:17241118

Ocular Purine Receptors as Drug Targets in the Eye.

PubMed

Jacobson, Kenneth A; Civan, Mortimer M

2016-10-01

Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A 1 , A 2A , and A 3 . An A 1 AR agonist is in clinical trials for glaucoma, A 2A AR reduces neuroinflammation, A 3 AR protects retinal ganglion cells from apoptosis, and both A 3 AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y 2 and P2Y 6 , lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases.

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.

PubMed

Wootten, Denise; Reynolds, Christopher A; Smith, Kevin J; Mobarec, Juan C; Koole, Cassandra; Savage, Emilia E; Pabreja, Kavita; Simms, John; Sridhar, Rohan; Furness, Sebastian G B; Liu, Mengjie; Thompson, Philip E; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M

2016-06-16

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

PubMed

Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi

2017-11-15

The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.

Dopamine modulation of transient receptor potential vanilloid type 1 (TRPV1) receptor in dorsal root ganglia neurons.

PubMed

Chakraborty, Saikat; Rebecchi, Mario; Kaczocha, Martin; Puopolo, Michelino

2016-03-15

The transient receptor potential vanilloid type 1 (TRPV1) receptor plays a key role in the modulation of nociceptor excitability. To address whether dopamine can modulate the activity of TRPV1 channels in nociceptive neurons, the effects of dopamine and dopamine receptor agonists were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27 μm) dorsal root ganglia (DRG) neurons. Dopamine or SKF 81297 (an agonist at D1/D5 receptors), caused inhibition of both inward and outward currents by ∼60% and ∼48%, respectively. The effect of SKF 81297 was reversed by SCH 23390 (an antagonist at D1/D5 receptors), confirming that it was mediated by activation of D1/D5 dopamine receptors. In contrast, quinpirole (an agonist at D2 receptors) had no significant effect on the capsaicin-activated current. Inhibition of the capsaicin-activated current by SKF 81297 was mediated by G protein coupled receptors (GPCRs), and highly dependent on external calcium. The inhibitory effect of SKF 81297 on the capsaicin-activated current was not affected when the protein kinase A (PKA) activity was blocked with H89, or when the protein kinase C (PKC) activity was blocked with bisindolylmaleimide II (BIM). In contrast, when the calcium-calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of SKF 81297 on the capsaicin-activated current was greatly reduced, suggesting that activation of D1/D5 dopamine receptors may be preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by dopamine in nociceptive neurons may represent a way for dopamine to modulate incoming noxious stimuli. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Ocular Purine Receptors as Drug Targets in the Eye

PubMed Central

Civan, Mortimer M.

2016-01-01

Abstract Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A1, A2A, and A3. An A1AR agonist is in clinical trials for glaucoma, A2AAR reduces neuroinflammation, A3AR protects retinal ganglion cells from apoptosis, and both A3AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y2 and P2Y6, lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases. PMID:27574786

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis

PubMed Central

Jenkinson, Karl M; Southwell, Bridget R; Furness, John B

1999-01-01

In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r.We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum.SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis.The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP.The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. PMID:10051129

Potential antipsychotic properties of central cannabinoid (CB1) receptor antagonists.

PubMed

Roser, Patrik; Vollenweider, Franz X; Kawohl, Wolfram

2010-03-01

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), the principal psychoactive constituent of the Cannabis sativa plant, and other agonists at the central cannabinoid (CB(1)) receptor may induce characteristic psychomotor effects, psychotic reactions and cognitive impairment resembling schizophrenia. These effects of Delta(9)-THC can be reduced in animal and human models of psychopathology by two exogenous cannabinoids, cannabidiol (CBD) and SR141716. CBD is the second most abundant constituent of Cannabis sativa that has weak partial antagonistic properties at the CB(1) receptor. CBD inhibits the reuptake and hydrolysis of anandamide, the most important endogenous CB(1) receptor agonist, and exhibits neuroprotective antioxidant activity. SR141716 is a potent and selective CB(1) receptor antagonist. Since both CBD and SR141716 can reverse many of the biochemical, physiological and behavioural effects of CB(1) receptor agonists, it has been proposed that both CBD and SR141716 have antipsychotic properties. Various experimental studies in animals, healthy human volunteers, and schizophrenic patients support this notion. Moreover, recent studies suggest that cannabinoids such as CBD and SR141716 have a pharmacological profile similar to that of atypical antipsychotic drugs. In this review, both preclinical and clinical studies investigating the potential antipsychotic effects of both CBD and SR141716 are presented together with the possible underlying mechanisms of action.

Design of Glucagon-Like Peptide-1 Receptor Agonist for Diabetes Mellitus from Traditional Chinese Medicine

PubMed Central

Tang, Hsin-Chieh; Chen, Calvin Yu-Chian

2014-01-01

Glucagon-like peptide-1 (GLP-1) is a promising target for diabetes mellitus (DM) therapy and reduces the occurrence of diabetes due to obesity. However, GLP-1 will be hydrolyzed soon by the enzyme dipeptidyl peptidase-4 (DPP-4). We tried to design small molecular drugs for GLP-1 receptor agonist from the world's largest traditional Chinese medicine (TCM) Database@Taiwan. According to docking results of virtual screening, we selected 2 TCM compounds, wenyujinoside and 28-deglucosylchikusetsusaponin IV, for further molecular dynamics (MD) simulation. GLP-1 was assigned as the control compound. Based on the results of root mean square deviation (RMSD), solvent accessible surface (SAS), mean square deviation (MSD), Gyrate, total energy, root mean square fluctuation (RMSF), matrices of smallest distance of residues, database of secondary structure assignment (DSSP), cluster analysis, and distance of H-bond, we concluded that all the 3 compounds could bind and activate GLP-1 receptor by computational simulation. Wenyujinoside and 28-deglucosylchikusetsusaponin IV were the TCM compounds that could be GLP-1 receptor agonists. PMID:24891870

Design of glucagon-like Peptide-1 receptor agonist for diabetes mellitus from traditional chinese medicine.

PubMed

Tang, Hsin-Chieh; Chen, Calvin Yu-Chian

2014-01-01

Glucagon-like peptide-1 (GLP-1) is a promising target for diabetes mellitus (DM) therapy and reduces the occurrence of diabetes due to obesity. However, GLP-1 will be hydrolyzed soon by the enzyme dipeptidyl peptidase-4 (DPP-4). We tried to design small molecular drugs for GLP-1 receptor agonist from the world's largest traditional Chinese medicine (TCM) Database@Taiwan. According to docking results of virtual screening, we selected 2 TCM compounds, wenyujinoside and 28-deglucosylchikusetsusaponin IV, for further molecular dynamics (MD) simulation. GLP-1 was assigned as the control compound. Based on the results of root mean square deviation (RMSD), solvent accessible surface (SAS), mean square deviation (MSD), Gyrate, total energy, root mean square fluctuation (RMSF), matrices of smallest distance of residues, database of secondary structure assignment (DSSP), cluster analysis, and distance of H-bond, we concluded that all the 3 compounds could bind and activate GLP-1 receptor by computational simulation. Wenyujinoside and 28-deglucosylchikusetsusaponin IV were the TCM compounds that could be GLP-1 receptor agonists.

A pharmacological study of NK1 and NK2 tachykinin receptor characteristics in the rat isolated urinary bladder.

PubMed Central

Hall, J. M.; Flowers, J. M.; Morton, I. K.

1992-01-01

1. We have estimated potencies of tachykinin receptor agonist and antagonist analogues in order to determine the recognition characteristics of tachykinin receptors mediating phasic contractile responses of the rat isolated urinary bladder in vitro. 2. The NK1-selective synthetic agonists, substance P methyl ester and GR73632, the synthetic NK2-selective agonists [beta-Ala8]-NKA(4-10) and GR64349, and the mammalian tachykinins, neurokinin A and neurokinin B, were assayed relative to substance P and were found to be approximately equipotent. The NK3-selective agonist, senktide, was inactive (10 microM). 3. Potencies of all these agonists were not significantly different (P > 0.05) when experiments were carried out in the presence of the neutral endopeptidase inhibitor, phosphoramidon, and the kininase II inhibitor, enalaprilat (both 1 microM). 4. The NK1-selective antagonist, GR82334, inhibited responses to substance P methyl ester in a competitive manner in the rat urinary bladder and the rat ileum, and also in the guinea-pig ileum. Markedly different pKB estimates were obtained in the rat bladder (6.38) and rat ileum (6.56) compared to the guinea-pig ileum (7.42). GR82334 (3 microM) was inactive against responses of the rat bladder to [beta-Ala8]-NKA(4-10). 5. The NK1-selective antagonist (+/-)-CP-96,345 also inhibited responses of the rat bladder and guinea-pig ileum to substance P methyl ester; however, in the rat bladder at 1 microM, this antagonist reversibly inhibited responses both to the NK2-selective agonist [beta-Ala8]-NKA(4-10) and to the muscarinic agonist carbachol (P < or = 0.01), thus showing evidence of some non-selective depressant actions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1282072

Modulatory effect of the 5-HT1A agonist buspirone and the mixed non-hallucinogenic 5-HT1A/2A agonist ergotamine on psilocybin-induced psychedelic experience.

PubMed

Pokorny, Thomas; Preller, Katrin H; Kraehenmann, Rainer; Vollenweider, Franz X

2016-04-01

The mixed serotonin (5-HT) 1A/2A/2B/2C/6/7 receptor agonist psilocybin dose-dependently induces an altered state of consciousness (ASC) that is characterized by changes in sensory perception, mood, thought, and the sense of self. The psychological effects of psilocybin are primarily mediated by 5-HT2A receptor activation. However, accumulating evidence suggests that 5-HT1A or an interaction between 5-HT1A and 5-HT2A receptors may contribute to the overall effects of psilocybin. Therefore, we used a double-blind, counterbalanced, within-subject design to investigate the modulatory effects of the partial 5-HT1A agonist buspirone (20mg p.o.) and the non-hallucinogenic 5-HT2A/1A agonist ergotamine (3mg p.o.) on psilocybin-induced (170 µg/kg p.o.) psychological effects in two groups (n=19, n=17) of healthy human subjects. Psychological effects were assessed using the Altered State of Consciousness (5D-ASC) rating scale. Buspirone significantly reduced the 5D-ASC main scale score for Visionary Restructuralization (VR) (p<0.001), which was mostly driven by a reduction of the VR item cluster scores for elementary and complex visual hallucinations. Further, buspirone also reduced the main scale score for Oceanic Boundlessness (OB) including derealisation and depersonalisation phenomena at a trend level (p=0.062), whereas ergotamine did not show any effects on the psilocybin-induced 5D-ASC main scale scores. The present finding demonstrates that buspirone exerts inhibitory effects on psilocybin-induced effects, presumably via 5-HT1A receptor activation, an interaction between 5-HT1A and 5-HT2A receptors, or both. The data suggest that the modulation of 5-HT1A receptor activity may be a useful target in the treatment of visual hallucinations in different psychiatric and neurological diseases. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

SGLT2 inhibitors or GLP-1 receptor agonists as second-line therapy in type 2 diabetes: patient selection and perspectives.

PubMed

Gurgle, Holly E; White, Karen; McAdam-Marx, Carrie

2016-01-01

Controversy exists regarding the selection of second-line therapy for patients with type 2 diabetes mellitus (T2DM) who are unable to achieve glycemic control with metformin therapy alone. Newer pharmacologic treatments for T2DM include glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter 2 inhibitors. Both the classes of medication are efficacious, exhibit positive effects on weight, and are associated with minimal risk of hypoglycemia. The purpose of this review is to compare the clinical trial and real-world effectiveness data of glucagon-like peptide-1 receptor agonists versus sodium-glucose cotransporter 2 inhibitors related to A1c reduction, weight loss, cost-effectiveness, cardiovascular outcomes, and safety in patients with T2DM. This review summarizes comparative evidence for providers who are determining which of the two classes may be the most appropriate for a specific patient.

Both substance P agonists and antagonists inhibit ion conductance through nicotinic acetylcholine receptors on PC12 cells.

PubMed

Eardley, D; McGee, R

1985-08-07

Substance P stimulates substance P receptors but also inhibits ion conductance through nicotinic acetylcholine receptors. Substance P analogs, classified as agonists or antagonists based on their actions on smooth muscle, were tested to determine if they also could act at nicotinic receptors on the pheochromocytoma, PC12. All of the analogs tested, [D-Pro2, D-Trp7,9]SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP, [pGlu5, MePhe8, Sar9]SP-(5-11), and [D-Pro4, D-Trp7,9,10]SP-(4-11), inhibited agonist-induced uptake of 86Rb+ through the nicotinic receptors at concentrations quite similar to those required for action at substance P receptors on smooth muscle. Thus, the chemical modifications in the analogs do not substantially alter their ability to inhibit nicotinic receptors.

Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

PubMed

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

PubMed Central

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

Glucagon-like peptide-1 receptor agonists versus insulin glargine for type 2 diabetes mellitus: A systematic review and meta-analysis of randomized controlled trials

PubMed Central

Li, Wei-Xin; Gou, Jian-Feng; Tian, Jin-Hui; Yan, Xiang; Yang, Lin

2010-01-01

Background: Glucagon-like peptide-1 (GLP-1) receptor agonists are a new class of hypoglycemic drugs, including exenatide, liraglutide, albiglutide, lixisenatide, and taspoglutide. Insulin glargine is a standard agent used to supplement basal insulin in type 2 diabetes mellitus (T2DM). Objective: The aim of this study was to review the efficacy and safety profiles of GLP-1 receptor agonists versus insulin glargine in type 2 diabetic patients who have not achieved treatment goals with oral hypoglycemic agents. Methods: The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and the database of ongoing trials were searched from inception through April 2010. Additional data were sought from relevant Web sites, the American Diabetes Association, reference lists of included trials and related (systematic) reviews, and industry. Randomized controlled trials (RCTs) were selected if they were ≥3 months in duration, compared GLP-1 receptor agonists with insulin glargine in patients with T2DM, and included ≥1 of the following outcomes: mortality, complications of T2DM, glycemie control, weight, lipids, blood pressure, adverse effects, and health-related quality of life. Quasirandomized controlled trials were excluded. The quality of the eligible studies was assessed on the basis of the following aspects: randomization procedure, allocation concealment, blinding, incomplete outcome data (intent-to-treat [ITT] analysis), selective outcome reporting, and publication bias. Results: A total of 410 citations were retrieved; 5 multicenter RCTs that met the inclusion criteria were identified. They were all open-label designs with an insulin glargine arm, predefined outcomes reported, and ITT analysis. One trial had an unclear randomization procedure and allocation concealment. Publication bias was not able to be determined. No data wete found with regard to mortality or diabetes-associated complications, and few data were found on quality of life. The results of the metaanalysis suggest that insulin glargine was significantly better in reducing the fasting blood glucose (mean difference [MD] [95% CI], 1.31 [1.04 to 1.58]; P < 0.001), but exhibits greater incidence of nocturnal hypoglycemia (risk ratio [RR] [95% CI], 0.40 [0.23 to 0.71]; P = 0.002) and influenza (RR [95% CI], 0.56 [0.32 to 0.98]; P = 0.04). GLP-1 receptor agonists are more conducive to reducing weight (MD [95% CI], −3.96 [−5.14 to -2.77]; P < 0.001), postprandial blood glucose (after breakfast, P < 0.001; after dinner, P < 0.001), and LDL-C (MD [95% CI], −0.18 [−0.28 to −0.08]; P < 0.001), but have significantly more gastrointestinal adverse effects (eg, nausea/ vomiting, P < 0.001). There were no significant differences between GLP-1 receptor agonists and insulin glargine in reducing glycosylated hemoglobin (HbA1c) levels (MD [95% CI], −0.03 [−0.13 to 0.08]) and the overall incidence of hypoglycemia (RR [95% CI], 0.69 [0.42 to 1.14]). Conclusions: Compared with insulin glargine, GLP-1 receptor agonists did not have a significant difference in regard to reducing HbA1c levels and they were significantly associated with decreased weight but increased gastrointestinal adverse events. It remains unclear whether GLP-1 receptor agonists influence mortality or diabetes-associated complications in patients with T2DM. More trials with longer follow-up are needed to determine the exact long-term efficacy and safety profiles of this new class of hypoglycemic drugs. PMID:24688145

Caged Naloxone Reveals Opioid Signaling Deactivation Kinetics

PubMed Central

Banghart, Matthew R.; Shah, Ruchir C.; Lavis, Luke D.

2013-01-01

The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis. PMID:23960100

Sigma-1 (σ1) Receptor in Memory and Neurodegenerative Diseases.

PubMed

Maurice, Tangui; Goguadze, Nino

2017-01-01

The sigma-1 (σ 1 ) receptor has been associated with regulation of intracellular Ca 2+ homeostasis, several cellular signaling pathways, and inter-organelle communication, in part through its chaperone activity. In vivo, agonists of the σ 1 receptor enhance brain plasticity, with particularly well-described impact on learning and memory. Under pathological conditions, σ 1 receptor agonists can induce cytoprotective responses. These protective responses comprise various complementary pathways that appear to be differentially engaged according to pathological mechanism. Recent studies have highlighted the efficacy of drugs that act through the σ 1 receptor to mitigate symptoms associated with neurodegenerative disorders with distinct mechanisms of pathogenesis. Here, we will review genetic and pharmacological evidence of σ 1 receptor engagement in learning and memory disorders, cognitive impairment, and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington's disease.

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

Differentiation of δ, μ, and κ opioid receptor agonists based on pharmacophore development and computed physicochemical properties

NASA Astrophysics Data System (ADS)

Filizola, Marta; Villar, Hugo O.; Loew, Gilda H.

2001-04-01

Compounds that bind with significant affinity to the opioid receptor types, δ, μ, and κ, with different combinations of activation and inhibition at these three receptors could be promising behaviorally selective agents. Working on this hypothesis, the chemical moieties common to three different sets of opioid receptor agonists with significant affinity for each of the three receptor types δ, μ, or κ were identified. Using a distance analysis approach, common geometric arrangements of these chemical moieties were found for selected δ, μ, or κ opioid agonists. The chemical and geometric commonalities among agonists at each opioid receptor type were then compared with a non-specific opioid recognition pharmacophore recently developed. The comparison provided identification of the additional requirements for activation of δ, μ, and κ opioid receptors. The distance analysis approach was able to clearly discriminate κ-agonists, while global molecular properties for all compounds were calculated to identify additional requirements for activation of δ and μ receptors. Comparisons of the combined geometric and physicochemical properties calculated for each of the three sets of agonists allowed the determination of unique requirements for activation of each of the three opioid receptors. These results can be used to improve the activation selectivity of known opioid agonists and as a guide for the identification of novel selective opioid ligands with potential therapeutic usefulness.

Melanocortin Antagonist Tetrapeptides with Minimal Agonist Activity at the Mouse Melanocortin-3 Receptor

PubMed Central

2014-01-01

The melanocortin system regulates many important functions in the body. There are five melanocortin G protein-coupled receptor subtypes known to date. Herein, we report a structure–activity relationship (SAR) study of a tetrapeptide lead discovered through a double substitution strategy at the melanocortin core His-Phe-Arg-Trp sequence. Several compounds were identified with micromolar agonist activity at the mouse melanocortin-1 (mMC1R) and mouse melanocortin-5 receptor (mMC5R) subtypes, weak antagonist activity at the mouse melanocortin-3 receptor (mMC3R), and potent antagonist activity at the mouse melanocortin-4 receptor (mMC4R). Two compounds (2 and 3) were nanomolar mMC4R antagonists with no mMC3R antagonist activity observed. Additionally, we identified three tetrapeptide MC3R antagonists (1, 6, and 7) that possess minimal mMC3R agonist activity only at 100 μM, not commonly observed for mMC3R/mMC4R antagonists. These novel molecular templates have the potential as molecular probes to better differentiate the roles of the centrally expressed MC3 and MC4 receptors. PMID:25699138

Agonist-induced modulation of inverse agonist efficacy at the beta 2-adrenergic receptor.

PubMed

Chidiac, P; Nouet, S; Bouvier, M

1996-09-01

Sustained stimulation of several G protein-coupled receptors is known to lead to a reduction in the signaling efficacy. This phenomenon, named agonist-induced desensitization, has been best studied for the beta 2-adrenergic receptor (AR) and is characterized by a decreased efficacy of beta-adrenergic agonists to stimulate the adenylyl cyclase activity. Recently, several beta-adrenergic ligands were found to inhibit the spontaneous agonist-independent activity of the beta 2AR. These compounds, termed inverse agonists, have different inhibitory efficacies, ranging from almost neutral antagonists to full inverse agonists. The current study was undertaken to determine whether, as is the case for agonists, desensitization can affect the efficacies of inverse agonists. Agonist-promoted desensitization of the human beta 2AR expressed in Sf9 cells potentiated the inhibitory actions of the inverse agonists, with the extent of the potentiation being inversely proportional to their intrinsic activity. For example, desensitization increased the inhibitory action of the weak inverse agonist labetalol by 29%, whereas inhibition of the spontaneous activity by the strong inverse agonist timolol was not enhanced by the desensitizing stimuli. Interestingly, dichloroisoproterenol acted stochastically as either a weak partial agonist or a weak inverse agonist in control conditions but always behaved as an inverse agonist after desensitization. These data demonstrate that like for agonists, the efficacies of inverse agonists can be modulated by a desensitizing treatment. Also, the data show that the initial state of the receptor can determine whether a ligand behaves as a partial agonist or an inverse agonist.

Interaction of prostanoid EP3 and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE2

PubMed Central

Jones, RL; Woodward, DF

2011-01-01

BACKGROUND AND PURPOSE Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E2 (DX-DM PGE2) on pig cerebral artery when used as a selective EP3 receptor agonist. This study investigated the selectivity profile of DX-DM PGE2, focusing on the interaction between its EP3 and TP (thromboxane A2-like) agonist activities. EXPERIMENTAL APPROACH Contraction of guinea-pig trachea (EP1 system) and aorta (EP3 and TP systems) was measured in conventional organ baths. KEY RESULTS Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE2 (EP3 agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE2 induced strong contraction, which on the basis of treatment with (DG)-3ap (EP3 antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP3 and TP receptors. EP3/TP self-synergism also accounted for contraction induced by PGF2α and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE2 also showed significant EP1 agonism on guinea-pig trachea as defined by the EP1 antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM. CONCLUSIONS AND IMPLICATIONS EP3/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP3 agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP3 system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed. PMID:20955363

Cholinergic agonists increase intracellular calcium concentration in guinea pig vestibular hair cells.

PubMed

Han, W; Zhang, S; Han, D; Jiang, S; Yang, W

2001-07-01

To better understand the cholinergic receptors in vestibular hair cells (VHC) and their subtypes, and to investigate the effects of cholinergic agonists on intracellular calcium concentration ([Ca2+]i) in guinea pig VHCs. VHCs were isolated from guinea pig crista ampullaris by enzymatic and mechanical methods. The effect of cholinergic agonists on [Ca2+]i was examined using laser scanning confocal microscopy and the Ca2+ sensitive dye Fluo-3. The results showed that the addition of acetylcholine (ACh) and carbachol (CCh), muscamic and nicotinic agonists, induced [Ca2+]i increases in all the VHCs, whereas acetylcholine bromide (ACh-Br), a nicotinic agonist, induced the [Ca2+]i increase in only a small percentage of VHCs. The ACh or CCh-induced Ca2+ response could be partially suppressed by atropine. In the presence of 0.1 mmol/L atropine, the amplitudes of ACh or CCh-induced [Ca2+]i responses became significantly smaller than those in atropine free medium (P < 0.01). The results suggest the existence of cholinergic receptors in guinea pig VHCs. It is the muscamic agonists rather than nicontic receptors that dominate [Ca2+]i variation. Atropine can suppress muscamic agonist-induced Ca2+ responses.

Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

PubMed

Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

1998-08-01

This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT1A receptors. This affected markedly the affinity ratios of certain compounds. For example, (+/-)-idazoxan was only 3.6-fold selective for h alpha2A versus h5-HT1A but 51-fold selective for r alpha2A versus r5-HT1A receptors. Conversely, yohimbine was tenfold selective for h alpha2A versus h5-HT1A adrenoceptors but 4.2-fold selective for r alpha2A versus r5-HT1A receptors. Nevertheless, both atipamezole and DMT were highly selective for both rat and human alpha2A versus rat or human 5-HT1A receptors. In conclusion, these data indicate that: (1) the agonist DMT and the antagonist atipamezole are the ligands of choice to distinguish alpha2-mediated from 5-HT1A-mediated actions, whilst several of the other compounds show only low or modest selectivity for alpha2A over 5-HT1A receptors; (2) caution should be exercised in experimental and clinical interpretation of the actions of traditionally employed alpha2 ligands, such as clonidine, yohimbine and (+/-)-idazoxan, which exhibit marked agonist activity at 5-HT1A receptors.

Suppression of PLCβ2 by Endotoxin Plays a Role in the Adenosine A2A Receptor-Mediated Switch of Macrophages from an Inflammatory to an Angiogenic Phenotype

PubMed Central

Grinberg, Stan; Hasko, Gyorgy; Wu, Dianqing; Leibovich, Samuel Joseph

2009-01-01

Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A2A receptor (A2AR) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A2ARs by TLR agonists, down-regulation of tumor necrosis factor α (TNFα) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)β1 and β2 expression in macrophages by de-stabilizing their mRNAs. The PLCβ inhibitor U73122 down-regulates TNFα expression by macrophages, and in the presence of A2AR agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A2AR agonists. Selective down-regulation of PLCβ2, but not PLCβ1, using small-interfering RNA resulted in increased VEGF expression in response to A2AR agonists, but did not suppress TNFα expression. Macrophages from PLCβ2−/− mice also expressed increased VEGF in response to A2AR agonists. LPS-mediated suppression of PLCβ1 and β2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 μg/mouse, i.p.) suppressed PLCβ1 and β2 expression in spleen, liver, and lung of wild-type but not MyD88−/− mice. These studies indicate that LPS suppresses PLCβ1 and β2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCβ2 plays an important role in switching M1 macrophages into an M2-like state. PMID:19850892

Comparison of hippocampal G protein activation by 5-HT(1A) receptor agonists and the atypical antipsychotics clozapine and S16924.

PubMed

Newman-Tancredi, A; Rivet, J-M; Cussac, D; Touzard, M; Chaput, C; Marini, L; Millan, M J

2003-09-01

This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT(1A) receptors in the hippocampus. These data support a role of postsynaptic 5-HT(1A) receptors in the functional profiles of certain antipsychotic agents.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

PubMed

Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

2001-04-13

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or beta(2)-ARS:

Cholinergic Neurotransmission: Function and Dysfunction, International Cholinergic Symposium (8th) Held at Montreal (Quebec) on 26-30 July 1992

DTIC Science & Technology

1992-12-31

receptor were decreased. In the presence of nicotine 1.0pM, the Kd values of rat cerebral muscarinic receptor bound with its agonist P3H] oxotremorine -M...inhibitory effects of GTPrS on [1 3H] oxotremorine -M binding were potentiated.It is suggsted that the binding properties of brain muscarinic receptor...interval) the dose-response curves of M-agonists arecoline and oxotremorine for producing salivation shifted leftward. Above demonstrated phenomena

TRPV1 channels in the nucleus of the solitary tract mediate thermal prolongation of the LCR in decerebrate piglets

PubMed Central

Xia, Luxi; Bartlett, Donald; Leiter, J.C.

2011-01-01

Elevating body temperature or just the temperature of the dorsal medulla by approximately 2 °C prolongs the laryngeal chemoreflex (LCR) in decerebrate neonatal piglets. We tested the hypothesis that transient receptor potential vanilloid 1 (TRPV1) receptors in the nucleus of the solitary tract (NTS) mediate thermal prolongation of the LCR. We studied the effect of a selective TRPV1 receptor antagonist on thermal prolongation of the LCR, and we tested the effect of a TRPV1 agonist on the duration of the LCR under normothermic conditions. We studied 37 decerebrate neonatal piglets between the ages of post-natal days 4 and 7. The TRPV1 receptor antagonist, 5−iodoresiniferatoxin (65 microM/L in 100 nL), blocked thermal prolongation of the LCR when injected bilaterally into the region of the NTS. The TRPV1 agonist, resiniferatoxin (0.65-1.0 mM/L in 100 nL), prolonged the LCR after bilateral injection into the NTS even when the body temperature of each piglet was normal. The effect of the TRPV1 agonists could be blocked by treatment with the GABAA receptor antagonist, bicuculline, whether given intravenously (0.3 mg/kg) or focally injected bilaterally into the NTS (10 mM in 100nL). We conclude that TRPV1 receptors in the NTS mediate thermal prolongation of the LCR. PMID:21276877

Stimulants as Specific Inducers of Dopamine-Independent σ Agonist Self-Administration in Rats

PubMed Central

Hiranita, Takato; Soto, Paul L.; Tanda, Gianluigi; Kopajtic, Theresa A.

2013-01-01

A previous study showed that cocaine self-administration induced dopamine-independent reinforcing effects of σ agonists mediated by their selective actions at σ1 receptors (σ1Rs), which are intracellularly mobile chaperone proteins implicated in abuse-related effects of stimulants. The present study assessed whether the induction was specific to self-administration of cocaine. Rats were trained to self-administer the dopamine releaser, d-methamphetamine (0.01–0.32 mg/kg per injection), the μ-opioid receptor agonist, heroin (0.001–0.032 mg/kg per injection), and the noncompetitive N-methyl-d-aspartate receptor/channel antagonist ketamine (0.032–1.0 mg/kg per injection). As with cocaine, self-administration of d-methamphetamine induced reinforcing effects of the selective σ1R agonists PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate hydrochloride] and (+)-pentazocine (0.032–1.0 mg/kg per injection, each). In contrast, neither self-administration of heroin nor ketamine induced PRE-084 or (+)-pentazocine (0.032–10 mg/kg per injection, each) self-administration. Although the σ1R agonists did not maintain responding in subjects with histories of heroin or ketamine self-administration, substitution for those drugs was obtained with appropriate agonists (e.g., remifentanil, 0.1–3.2 µg/kg per injection, for heroin and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+)-MK 801; dizocilpine), 0.32–10.0 µg/kg per injection, for ketamine). The σR antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008; 1.0–10 mg/kg) dose-dependently blocked PRE-084 self-administration but was inactive against d-methamphetamine, heroin, and ketamine. In contrast, PRE-084 self-administration was affected neither by the dopamine receptor antagonist (+)-butaclamol (10–100 μg/kg) nor by the opioid antagonist (−)-naltrexone (1.0–10 mg/kg), whereas these antagonists were active against d-methamphetamine and heroin self-administration, respectively. The results indicate that experience specifically with indirect-acting dopamine agonists induces reinforcing effects of previously inactive σ1R agonists. It is further suggested that induced σ1R reinforcing mechanisms may play an essential role in treatment-resistant stimulant abuse, suggesting new approaches for the development of effective medications for its treatment. PMID:23908387

Stimulants as specific inducers of dopamine-independent σ agonist self-administration in rats.

PubMed

Hiranita, Takato; Soto, Paul L; Tanda, Gianluigi; Kopajtic, Theresa A; Katz, Jonathan L

2013-10-01

A previous study showed that cocaine self-administration induced dopamine-independent reinforcing effects of σ agonists mediated by their selective actions at σ1 receptors (σ1Rs), which are intracellularly mobile chaperone proteins implicated in abuse-related effects of stimulants. The present study assessed whether the induction was specific to self-administration of cocaine. Rats were trained to self-administer the dopamine releaser, d-methamphetamine (0.01-0.32 mg/kg per injection), the μ-opioid receptor agonist, heroin (0.001-0.032 mg/kg per injection), and the noncompetitive N-methyl-d-aspartate receptor/channel antagonist ketamine (0.032-1.0 mg/kg per injection). As with cocaine, self-administration of d-methamphetamine induced reinforcing effects of the selective σ1R agonists PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate hydrochloride] and (+)-pentazocine (0.032-1.0 mg/kg per injection, each). In contrast, neither self-administration of heroin nor ketamine induced PRE-084 or (+)-pentazocine (0.032-10 mg/kg per injection, each) self-administration. Although the σ1R agonists did not maintain responding in subjects with histories of heroin or ketamine self-administration, substitution for those drugs was obtained with appropriate agonists (e.g., remifentanil, 0.1-3.2 µg/kg per injection, for heroin and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+)-MK 801; dizocilpine), 0.32-10.0 µg/kg per injection, for ketamine). The σR antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008; 1.0-10 mg/kg) dose-dependently blocked PRE-084 self-administration but was inactive against d-methamphetamine, heroin, and ketamine. In contrast, PRE-084 self-administration was affected neither by the dopamine receptor antagonist (+)-butaclamol (10-100 μg/kg) nor by the opioid antagonist (-)-naltrexone (1.0-10 mg/kg), whereas these antagonists were active against d-methamphetamine and heroin self-administration, respectively. The results indicate that experience specifically with indirect-acting dopamine agonists induces reinforcing effects of previously inactive σ1R agonists. It is further suggested that induced σ1R reinforcing mechanisms may play an essential role in treatment-resistant stimulant abuse, suggesting new approaches for the development of effective medications for its treatment.

Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes.

PubMed

Teitelbaum, A P; Silve, C M; Nyiredy, K O; Arnaud, C D

1986-02-01

Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest that the attenuation of PTH stimulation of cAMP production in treated bone cells may be, at least in part, due to receptor-mediated endocytosis of the hormone.

Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

PubMed

Monn, James A; Prieto, Lourdes; Taboada, Lorena; Hao, Junliang; Reinhard, Matthew R; Henry, Steven S; Beadle, Christopher D; Walton, Lesley; Man, Teresa; Rudyk, Helene; Clark, Barry; Tupper, David; Baker, S Richard; Lamas, Carlos; Montero, Carlos; Marcos, Alicia; Blanco, Jaime; Bures, Mark; Clawson, David K; Atwell, Shane; Lu, Frances; Wang, Jing; Russell, Marijane; Heinz, Beverly A; Wang, Xushan; Carter, Joan H; Getman, Brian G; Catlow, John T; Swanson, Steven; Johnson, Bryan G; Shaw, David B; McKinzie, David L

2015-09-24

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

PubMed

Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

PubMed

Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

2015-04-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening

PubMed Central

Law, Wenjing; Wuescher, Leah M.; Ortega, Amanda; Hapiak, Vera M.; Komuniecki, Patricia R.; Komuniecki, Richard

2015-01-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets. PMID:25928899

A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor

PubMed Central

Kaji, Mark D; Kwaka, Ariel; Callanan, Micah K; Nusrat, Humza; Desaulniers, Jean-Paul; Forrester, Sean G

2015-01-01

Background and Purpose Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. Experimental Approach We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. Key Results The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(−)-4-amino-3-hydroxybutyric acid [R(−)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(−)- and S(+)-GABOB. Conclusions and Implications The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes. PMID:25850584

AMP Is an Adenosine A1 Receptor Agonist*

PubMed Central

Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

2012-01-01

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

AMP is an adenosine A1 receptor agonist.

PubMed

Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

2012-02-17

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

Cell-Free Expression, Purification, and Characterization of the Functional β2-Adrenergic Receptor for Multianalyte Detection of β-Agonists.

PubMed

Wang, Jian; Liu, Yuan; Zhang, Junhua; Han, Zhengzheng; Wang, Wei; Liu, Yang; Wei, Dong; Huang, Wei

2017-11-01

Large-scale expression of β 2 -adrenergic receptor (β 2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β 2 -AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β 2 -AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β 2 -AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β 2 -AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.

Anticonvulsant activity of a mGlu(4alpha) receptor selective agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid.

PubMed

Chapman, A G; Talebi, A; Yip, P K; Meldrum, B S

2001-07-20

The metabotropic Group III agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), selective for the mGlu(4alpha) receptor, suppresses sound-induced seizures in DBA/2 mice following its intracerebroventricular (i.c.v.) administration (ED(50) 5.6 [2.9-10.7], nmol i.c.v., 15 min, clonic phase) and in genetically epilepsy-prone (GEP) rats following focal administration into the inferior colliculus (ED(50) 0.08 [0.01-0.50], nmol, 60 min, clonic phase). ACPT-1 also protects against clonic seizures induced in DBA/2 mice by the Group I agonist, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) (ED(50) 0.60 [0.29-1.2], nmol i.c.v.) and by the Group III antagonist, (RS)-alpha-methylserine-O-phosphate (MSOP) (ED(50) 49.3 [37.9-64.1], nmol i.c.v.). Another Group III agonist, (RS)-4-phosphonophenyl-glycine (PPG), preferentially activating the mGlu(8) receptor, previously shown to protect against sound-induced seizures in DBA/2 mice and GEP rats, also protects against seizures induced in DBA/2 by 3,5-DHPG (ED(50) 3.7 [2.4-5.7], nmol i.c.v.) and by the Group III antagonist, MSOP (ED(50) 40.2 [21.0-77.0], nmol i.c.v.). At very high doses (500 nmol i.c.v. and above), Group III antagonists have pro-convulsant and convulsant activity. The anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(4) receptor agonist ACPT-1, is partially reversed by the co-administration of the Group III antagonists, MSOP, (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) or (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), in the 20-50 nmol dose range. At doses of 50-200 nmol, MPPG and MAP4 cause further reversal of the ACPT-1 anticonvulsant protection, while the MSOP effect on ACPT-1 protection is abolished at higher doses. In contrast, the anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(8) receptor agonist PPG, is not significantly affected by the co-administration of the same Group III antagonists, MSOP, MPPG or MAP4. We conclude that activation of either mGlu(4alpha) or mGlu(8) receptors confer anticonvulsant protection in DBA/2 mice. Furthermore, the metabotropic Group III receptor antagonists, MSOP, MPPG, and MAP4 appear to be functionally selective for the mGlu(4) receptor in this system.

GPCRs as potential therapeutic targets in preeclampsia

PubMed Central

McGuane, JT; Conrad, KP

2012-01-01

Preeclampsia is an important obstetric complication that arises in 5% of women after the 20th week of gestation, for which there is no specific therapy and no cure. Although much of the recent investigation in this field has focused on soluble forms of the angiogenic membrane receptor tyrosine kinase Flt1 and the transforming growth factor β co-receptor Endoglin, there is significant clinical potential for several GPCR targets and their agonists or antagonists in preeclampsia. In this review, we discuss several of the most promising candidates in this category, including calcitonin receptor-like receptor / receptor activity modifying protein 1 complexes, the angiotensin AT1, 2 and Mas receptors, and the relaxin receptor RXFP1. We also address some of the controversies surrounding the roles and therapeutic potential of these GPCRs and their (ant)agonists in preeclampsia. PMID:23144646

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

PubMed Central

Giuliani, D; Mioni, C; Bazzani, C; Zaffe, D; Botticelli, A R; Capolongo, S; Sabba, A; Galantucci, M; Iannone, A; Grieco, P; Novellino, E; Colombo, G; Tomasi, A; Catania, A; Guarini, S

2007-01-01

Background and purpose: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Experimental approach: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP-α-MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock. PMID:17245369

Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression.

PubMed

Strekalova, Elena; Malin, Dmitry; Rajanala, Harisha; Cryns, Vincent L

2017-06-01

Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists. Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC. Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model. Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

CJ-1639: A Potent and Highly Selective Dopamine D3 Receptor Full Agonist.

PubMed

Chen, Jianyong; Collins, Gregory T; Levant, Beth; Woods, James; Deschamps, Jeffrey R; Wang, Shaomeng

2011-08-11

We have identified several ligands with high binding affinities to the dopamine D3 receptor and excellent selectivity over the D2 and D1 receptors. CJ-1639 (17) binds to the D3 receptor with a K(i) value of 0.50 nM and displays a selectivity of >5,000 times over D2 and D1 receptors in binding assays using dopamine receptors expressed in the native rat brain tissues. CJ-1639 binds to human D3 receptor with a K(i) value of 3.61 nM and displays over >1000-fold selectivity over human D1 and D2 receptors. CJ-1639 is active at 0.01 mg/kg at the dopamine D3 receptor in the rat and only starts to show a modest D2 activity at doses as high as 10 mg/kg. CJ-1639 is the most potent and selective D3 full agonist reported to date.

Analogues of doxanthrine reveal differences between the dopamine D 1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[a]phenanthridines

USGS Publications Warehouse

Cueva, J.P.; Chemel, B.R.; Juncosa, J.I.; Lill, M.A.; Watts, V.J.; Nichols, D.E.

2012-01-01

Efforts to develop selective agonists for dopamine D 1-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric ??-phenyldopamine-type full agonist ligands that display selectivity and potency at D 1-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D 1- and D 2-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D 1-like receptor binding, suggesting important differences between the interactions of these ligands with the D 1 receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor. ?? 2011 Elsevier Ltd. All rights reserved.

MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

PubMed

Rosso, Jean-Pierre; Schwarz, Jürgen R; Diaz-Bustamante, Marcelo; Céard, Brigitte; Gutiérrez, José M; Kneussel, Matthias; Pongs, Olaf; Bosmans, Frank; Bougis, Pierre E

2015-02-24

GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α(+)/β(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

Constitutive activity of the δ-opioid receptor expressed in C6 glioma cells: identification of non-peptide δ-inverse agonists

PubMed Central

Neilan, Claire L; Akil, Huda; Woods, James H; Traynor, John R

1999-01-01

G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ-(OP1) receptor (C6δ), or the cloned μ-(OP3) opioid receptor (C6μ).Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPγS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6δ cells the level of basal [35S]-GTPγS binding was reduced by 51.9±6.1 fmols mg−1 protein, whereas in C6μ and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg−1 protein respectively.The δ-antagonists N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as δ-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPγS binding in C6μ or C6 wild-type cell membranes.Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.These data confirm the presence of constitutive activity associated with the δ-opioid receptor and identify three novel, non-peptide, δ-opioid inverse agonists. PMID:10516632

A High-Throughput Screening Method to Identify Potential Pesticides for Mosquito Control

DTIC Science & Technology

2009-01-01

receptor agonists 5 Imidacloprid Nicotinic acetylcholine receptor agonist/antagonists 4 Diazinon Acetylcholinesterase inhibitors (organophosphates) 1B...0.84) 1.50 Spinosad 3.9 101 (3.6 101Ð4.1 101) 6.3 101 (5.5 101Ð7.9 101) 7.82 (1.33) 2.95 Imidacloprid 3.7 101 (2.9 101Ð4.5 101...pesticides (pyridaben, hydramethylnon, imidacloprid , diazinon, and indoxacarb) were moder- ately active against Þrst-instar larvae,withLC50 values of

Muscarinic and nicotinic acetylcholine receptor agonists: current scenario in Alzheimer's disease therapy.

PubMed

Verma, Stuti; Kumar, Ashwini; Tripathi, Timir; Kumar, Awanish

2018-04-16

Alzheimer's disease (AD) has become the primary cause of dementia. It shows a progressive cognitive dysfunction with degenerating neurons. Acetylcholine receptors (AChRs) propagate the cognitive ability and it consists of two primary members namely muscarinic (mAChRs) and nicotinic receptors (nAChRs). Where mAChRs is G-protein coupled receptor, (nAChRs) are ligand-gated ion channels. The conventional therapeutic regimen for AD consists of three acetylcholinestearse inhibitors while a single NMDA receptor antagonist. Researchers around the globe are developing new and modifying the existing AChRs agonists to develop lead candidates with lower risk to benefit ratio where benefits clearly outweigh the adverse events. We have searched PubMed, MEDLINE, Google scholar, Science Direct and, Web of Science with keywords "Muscarinic/Nicotinic acetylcholine receptor, agonists and, AD". The literature search included articles written in English. Scientific relevance for clinical studies, basic science studies is eligibility criteria for articles referred in this paper. M1 is the primary muscarinic subtype while α7 is the primary nAChR subtype that is responsible for cognition and memory and these two have been the major recent experimental targets for mAChR agonist strategy. The last cholinergic receptor agonist to enter phase 3 trial was EVP-6124 (Enceniclin) but was withdrawn due to severe gastrointestinal adverse effects. We aim to present an overview of the efforts and achievements in targeting Muscarinic and Nicotinic acetylcholine receptor in the current review for development of better AD therapeutics. © 2018 Royal Pharmaceutical Society.

PYY3–36 and Oxyntomodulin Can Be Additive in Their Effect on Food Intake in Overweight and Obese Humans

PubMed Central

Field, Benjamin C.T.; Wren, Alison M.; Peters, Veronique; Baynes, Kevin C.R.; Martin, Niamh M.; Patterson, Michael; Alsaraf, Sara; Amber, Vian; Wynne, Katie; Ghatei, Mohammad A.; Bloom, Stephen R.

2010-01-01

OBJECTIVE Peptide YY3–36 (PYY3–36), a Y2 receptor agonist, and oxyntomodulin, a glucagon-like peptide 1 (GLP-1) receptor agonist, are cosecreted by intestinal L-cells after each meal. Separately each hormone acts as an endogenous satiety signal and reduces appetite in humans when infused intravenously. The aim of the current study was to investigate whether the anorectic effects of PYY3–36 and oxyntomodulin can be additive. RESEARCH DESIGN AND METHODS Twelve overweight or obese human volunteers underwent a randomized, double-blinded, placebo-controlled study. An ad libitum test meal was used to measure energy intake during intravenous infusions of either PYY3–36 or oxyntomodulin or combined PYY3–36/oxyntomodulin. RESULTS Energy intake during coadministration of PYY3–36 and oxyntomodulin was reduced by 42.7% in comparison with the saline control and was significantly lower than that during infusions of either hormone alone. CONCLUSIONS The anorectic effects of PYY3–36 and oxyntomodulin can be additive in overweight and obese humans. Coadministration of Y2 receptor agonists and GLP-1 receptor agonists may be a useful treatment strategy for obesity. PMID:20357366

Toward an understanding of agonist binding to human Orexin-1 and Orexin-2 receptors with G-protein-coupled receptor modeling and site-directed mutagenesis.

PubMed

Heifetz, Alexander; Barker, Oliver; Morris, G Benjamin; Law, Richard J; Slack, Mark; Biggin, Philip C

2013-11-19

The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a molecular dynamics simulation and to help distinguish between different GPCR substates. The results demonstrate how these newly developed methods of structural assessment for GPCRs can be used to provide a working model of neuropeptide-Orexin receptor interaction.

Design and synthesis of a novel series of [1-(4-hydroxy-benzyl)-1H-indol-5-yloxy]-acetic acid compounds as potent, selective, thyroid hormone receptor β agonists.

PubMed

Burkholder, Timothy P; Cunningham, Brian E; Clayton, Joshua R; Lander, Peter A; Brown, Matthew L; Doti, Robert A; Durst, Gregory L; Montrose-Rafizadeh, Chahrzad; King, Constance; Osborne, Harold E; Amos, Robert M; Zink, Richard W; Stramm, Lawrence E; Burris, Thomas P; Cardona, Guemalli; Konkol, Debra L; Reidy, Charles; Christe, Michael E; Genin, Michael J

2015-04-01

The design, synthesis, and structure activity relationships for a novel series of indoles as potent, selective, thyroid hormone receptor β (TRβ) agonists is described. Compounds with >50× binding selectivity for TRβ over TRα were generated and evaluation of compound 1c from this series in a model of dyslipidemia demonstrated positive effects on plasma lipid endpoints in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

mGluR2/3 agonist LY379268 rescues NMDA and GABAA receptor level deficits induced in a two-hit mouse model of schizophrenia.

PubMed

Engel, Martin; Snikeris, Peta; Matosin, Natalie; Newell, Kelly Anne; Huang, Xu-Feng; Frank, Elisabeth

2016-04-01

An imbalance of excitatory and inhibitory neurotransmission underlies the glutamate hypothesis of schizophrenia. Agonists of group II metabotropic glutamate receptors, mGluR2/3, have been proposed as novel therapeutic agents to correct this imbalance. However, the influence of mGluR2/3 activity on excitatory and inhibitory neurotransmitter receptors has not been explored. We aimed to investigate the ability of a novel mGluR2/3 agonist, LY379268, to modulate the availability of the excitatory N-methyl-D-aspartate receptor (NMDA-R) and the inhibitory gamma-aminobutyrate-A receptor (GABAA-R), in a two-hit mouse model of schizophrenia. Wild type (WT) and heterozygous neuregulin 1 transmembrane domain mutant mice (NRG1 HET) were treated daily with phencyclidine (10 mg/kg ip) or saline for 14 days. After a 14-day washout, an acute dose of the mGluR2/3 agonist LY379268 (3 mg/kg), olanzapine (antipsychotic drug comparison, 1.5 mg/kg), or saline was administered. NMDA-R and GABAA-R binding densities were examined by receptor autoradiography in several schizophrenia-relevant brain regions. In both WT and NRG1 HET mice, phencyclidine treatment significantly reduced NMDA-R and GABAA-R binding density in the prefrontal cortex, hippocampus, and nucleus accumbens. Acute treatment with LY379268 restored NMDA-R and GABAA-R levels in the two-hit mouse model comparable to olanzapine. We demonstrate that the mGluR2/3 agonist LY379268 restores excitatory and inhibitory deficits with similar efficiency as olanzapine in our two-hit schizophrenia mouse model. This study significantly contributes to our understanding of the mechanisms underlying the therapeutic effects of LY379268 and supports the use of agents aimed at mGluR2/3.

Insights into the structure and pharmacology of the human trace amine-associated receptor 1 (hTAAR1): homology modelling and docking studies.

PubMed

Cichero, Elena; Espinoza, Stefano; Gainetdinov, Raul R; Brasili, Livio; Fossa, Paola

2013-04-01

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that belongs to the family of TAAR receptors and responds to a class of compounds called trace amines, such as β-phenylethylamine (β-PEA) and 3-iodothyronamine (T(1)AM). The receptor is known to have a very rich pharmacology and could be also activated by other classes of compounds, including adrenergic and serotonergic ligands. It is expected that targeting TAAR1 could provide a novel pharmacological approach to correct monoaminergic dysfunctions found in several brain disorders, such as schizophrenia, depression, attention deficit hyperactivity disorder and Parkinson's disease. Only recently, the first selective TAAR1 agonist RO5166017 has been identified. To explore the molecular mechanisms of protein-agonist interaction and speed up the identification of new chemical entities acting on this biomolecular target, we derived a homology model for the hTAAR1. The putative protein-binding site has been explored by comparing the hTAAR1 model with the β(2)-adrenoreceptor binding site, available by X-ray crystallization studies, and with the homology modelled 5HT(1A) receptor. The obtained results, in tandem with docking studies performed with RO5166017, β-PEA and T(1)AM, provided an opportunity to reasonably identify the hTAAR1 key residues involved in ligand recognition and thus define important starting points to design new agonists. © 2012 John Wiley & Sons A/S.

Characterization of the EP receptor types that mediate longitudinal smooth muscle contraction of human colon, mouse colon and mouse ileum.

PubMed

Fairbrother, S E; Smith, J E; Borman, R A; Cox, H M

2011-08-01

Prostaglandin E(2) (PGE(2) ) is an inflammatory mediator implicated in several gastrointestinal pathologies that affect normal intestinal transit. The aim was to establish the contribution of the four EP receptor types (EP(1-4) ), in human colon, that mediate PGE(2) -induced longitudinal smooth muscle contraction. Changes in isometric muscle tension of human colon, mouse colon and mouse ileum were measured in organ baths in response to receptor-specific agonists and antagonists. In addition, lidocaine was used to block neurogenic activity to investigate whether EP receptors were pre- or post-junctional. PGE(2) contracted longitudinal muscle from human and mouse colon and mouse ileum. These contractions were inhibited by the EP(1) receptor antagonist, EP(1) A in human colon, whereas a combination of EP(1) A and the EP(3) antagonist, L798106 inhibited agonist responses in both mouse preparations. The EP(3) agonist, sulprostone also increased muscle tension in both mouse tissues, and these responses were inhibited by lidocaine in the colon but not in the ileum. Although PGE(2) consistently contracted all three muscle preparations, butaprost decreased tension by activating smooth muscle EP(2) receptors in both colonic tissues. Alternatively, in mouse ileum, butaprost responses were lidocaine-sensitive, suggesting that it was activating prejunctional EP(2) receptors on inhibitory motor neurons. Conversely, EP(4) receptors were not functional in all the intestinal muscle preparations tested. PGE(2) -induced contraction of longitudinal smooth muscle is mediated by EP(1) receptors in human colon and by a combination of EP(1) and EP(3) receptors in mouse intestine, whereas EP(2) receptors modulate relaxation in all three preparations. © 2011 Blackwell Publishing Ltd.

Effects of oxytocin on serotonin 1B agonist-induced autism-like behavior in mice.

PubMed

Lawson, Sarah K; Gray, Andrew C; Woehrle, Nancy S

2016-11-01

Social impairments in autism remain poorly understood and without approved pharmacotherapies. Novel animals models are needed to elucidate mechanisms and evaluate novel treatments for the social deficits in autism. Recently, serotonin 1B receptor (5-HT1B) agonist challenge in mice was shown to induce autism-like behaviors including perseveration, reduced prepulse inhibition, and delayed alternation deficits. However, the effects of 5-HT1B agonists on autism-related social behaviors in mice remain unknown. Here, we examine the effects of 5-HT1B agonist challenge on sociability and preference for social novelty in mice. We also examine the effects of 5-HT1B agonist treatment on average rearing duration, a putative rodent measure of non-selective attention. Non-selective attention is an associated feature of autism that is also not well understood. We show that 5-HT1B receptor activation reduces sociability, preference for social novelty, and rearing in mice. In addition, we examine the ability of oxytocin, an off-label treatment for the social impairments in autism, to reverse 5-HT1B agonist-induced social and attention deficits in mice. We show that oxytocin restores social novelty preference in mice treated with a 5-HT1B agonist. We also show that oxytocin attenuates 5-HT1B agonist-induced sociability and rearing deficits in mice. Our results suggest that 5-HT1B agonist challenge provides a useful pharmacological mouse model for aspects of autism, and implicate 5-HT1B in autism social and attention deficits. Moreover, our findings suggest that oxytocin may treat the social deficits in autism through a mechanism involving 5-HT1B. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

PubMed Central

Nürnberg, Daniela; Grüters, Annette; Führer-Sakel, Dagmar; Krude, Heiko; Köhrle, Josef; Schöneberg, Torsten; Biebermann, Heike

2011-01-01

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes. PMID:22073124

Glucagon-like peptide-1 receptor agonists and heart failure in type 2 diabetes: systematic review and meta-analysis of randomized and observational studies.

PubMed

Li, Ling; Li, Sheyu; Liu, Jiali; Deng, Ke; Busse, Jason W; Vandvik, Per Olav; Wong, Evelyn; Sohani, Zahra N; Bala, Malgorzata M; Rios, Lorena P; Malaga, German; Ebrahim, Shanil; Shen, Jiantong; Zhang, Longhao; Zhao, Pujing; Chen, Qunfei; Wang, Yingqiang; Guyatt, Gordon H; Sun, Xin

2016-05-11

The effect of glucagon-like peptide-1(GLP-1) receptor agonists on heart failure remains uncertain. We therefore conducted a systematic review to assess the possible impact of GLP-1 agonists on heart failure or hospitalization for heart failure in patients with type 2 diabetes. We searched MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL) and ClinicalTrials.gov to identify randomized controlled trials (RCTs) and observational studies that addressed the effect of GLP-1 receptor agonists in adults with type 2 diabetes, and explicitly reported heart failure or hospitalization for heart failure. Two paired reviewers screened reports, collected data, and assessed the risk of bias. We pooled data from RCTs and observational studies separately, and used the GRADE approach to rate the quality of evidence. We identified 25 studies that were eligible for our review; 21 RCTs (n = 18,270) and 4 observational studies (n = 111,029). Low quality evidence from 20 RCTs suggested, if anything, a lower incidence of heart failure between GLP-1 agonists versus control (17/7,441 vs. 19/4,317; odds ratio (OR) 0.62, 95 % confidence interval (CI) 0.31 to 1.22; risk difference (RD) 19 fewer, 95 % CI 34 fewer to 11 more per 1000 over 5 years). Three cohort studies comparing GLP-1 agonists to alternative agents provided very low quality evidence that GLP-1 agonists do not increase the incidence of heart failure. One RCT provided moderate quality evidence that GLP-1 agonists were not associated with hospitalization for heart failure (lixisenatide vs placebo: 122/3,034 vs. 127/3,034; adjusted hazard ratio 0.96, 95 % CI 0.75 to 1.23; RD 4 fewer, 95 % CI 25 fewer to 23 more per 1000 over 5 years) and a case-control study provided very low quality evidence also suggesting no association (GLP-1 agonists vs. other anti-hyperglycemic drugs: 1118 cases and 17,626 controls, adjusted OR 0.67, 95 % CI 0.32 to 1.42). The current evidence suggests that GLP-1 agonists do not increase the risk of heart failure or hospitalization for heart failure among patients with type 2 diabetes.

[Effect of 5-HT1A receptors in the hippocampal DG on active avoidance learning in rats].

PubMed

Jiang, Feng-ze; Lv, Jing; Wang, Dan; Jiang, Hai-ying; Li, Ying-shun; Jin, Qing-hua

2015-01-01

To investigate the effects of serotonin (5-HTIA) receptors in the hippocampal dentate gyrus (DG) on active avoidance learning in rats. Totally 36 SD rats were randomly divided into control group, antagonist group and agonist group(n = 12). Active avoidance learning ability of rats was assessed by the shuttle box. The extracellular concentrations of 5-HT in the DG during active avoidance conditioned reflex were measured by microdialysis and high performance liquid chromatography (HPLC) techniques. Then the antagonist (WAY-100635) or agonist (8-OH-DPAT) of the 5-HT1A receptors were microinjected into the DG region, and the active avoidance learning was measured. (1) During the active avoidance learning, the concentration of 5-HT in the hippocampal DG was significantly increased in the extinction but not establishment in the conditioned reflex, which reached 164.90% ± 26.07% (P <0.05) of basal level. (2) The microinjection of WAY-100635 (an antagonist of 5-HT1A receptor) into the DG did not significantly affect the active avoidance learning. (3) The microinjection of 8-OH-DPAT(an agonist of 5-HT1A receptor) into the DG significantly facilitated the establishment process and inhibited the extinction process during active avoidance conditioned reflex. The data suggest that activation of 5-HT1A receptors in hipocampal DG may facilitate active avoidance learning and memory in rats.

Working memory span capacity improved by a D2 but not D1 receptor family agonist.

PubMed

Tarantino, Isadore S; Sharp, Richard F; Geyer, Mark A; Meves, Jessica M; Young, Jared W

2011-06-01

Patients with schizophrenia exhibit poor working memory (WM). Although several subcomponents of WM can be measured, evidence suggests the primary subcomponent affected in schizophrenia is span capacity (WMC). Indeed, the NIMH-funded MATRICS initiative recommended assaying the WMC when assessing the efficacy of a putative therapeutic for FDA approval. Although dopamine D1 receptor agonists improve delay-dependent memory in animals, evidence for improvements in WMC due to dopamine D1 receptor activation is limited. In contrast, the dopamine D2-family agonist bromocriptine improves WMC in humans. The radial arm maze (RAM) can be used to assess WMC, although complications due to ceiling effects or strategy confounds have limited its use. We describe a 12-arm RAM protocol designed to assess whether the dopamine D1-family agonist SKF 38393 (0, 1, 3, and 10 mg/kg) or bromocriptine (0, 1, 3, and 10 mg/kg) could improve WMC in C57BL/6N mice (n=12) in cross-over designs. WMC increased and strategy usage decreased with training. The dopamine D1 agonist SKF 38393 had no effect on WMC or long-term memory. Bromocriptine decreased WMC errors, without affecting long-term memory, consistent with human studies. These data confirm that WMC can be measured in mice and reveal drug effects that are consistent with reported effects in humans. Future research is warranted to identify the subtype of the D2-family of receptors responsible for the observed improvement in WMC. Finally, this RAM procedure may prove useful in developing animal models of deficient WMC to further assess putative treatments for the cognitive deficits in schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

A retinoic acid receptor β2 agonist reduces hepatic stellate cell activation in nonalcoholic fatty liver disease.

PubMed

Trasino, Steven E; Tang, Xiao-Han; Jessurun, Jose; Gudas, Lorraine J

2016-10-01

Hepatic stellate cells (HSCs) are an important cellular target for the development of novel pharmacological therapies to prevent and treat nonalcoholic fatty liver diseases (NAFLD). Using a high fat diet (HFD) model of NAFLD, we sought to determine if synthetic selective agonists for retinoic acid receptor β2 (RARβ2) and RARγ can mitigate HSC activation and HSC relevant signaling pathways during early stages of NAFLD, before the onset of liver injury. We demonstrate that the highly selective RARβ2 agonist, AC261066, can reduce the activation of HSCs, marked by decreased HSC expression of α-smooth muscle actin (α-SMA), in mice with HFD-induced NAFLD. Livers of HFD-fed mice treated with AC261066 exhibited reduced steatosis, oxidative stress, and expression of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFα), interleukin 1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1). Kupffer cell (macrophage) expression of transforming growth factor-β1 (TGF-β1), which plays a critical role in early HSC activation, was markedly reduced in AC261066-treated, HFD-fed mice. In contrast, HFD-fed mice treated with an RARγ agonist (CD1530) showed no decreases in steatosis, HSC activation, or Kupffer cell TGF-β1 levels. In conclusion, our data demonstrate that RARβ2 is an attractive target for development of NAFLD therapies. • Hepatic stellate cells (HSCs) are an important pharmacological target for the prevention of nonalcoholic fatty liver diseases (NAFLD). • Retinoids and retinoic acid receptors (RARs) possess favorable metabolic modulating properties. • We show that an agonist for retinoic acid receptor-β2 (RARβ2), but not RARγ, mitigates HSC activation and NAFLD.

PWZ-029, A COMPOUND WITH MODERATE INVERSE AGONIST FUNCTIONAL SELECTIVITY AT GABAA RECEPTORS CONTAINING α5 SUBUNITS, IMPROVES PASSIVE, BUT NOT ACTIVE, AVOIDANCE LEARNING IN RATS

PubMed Central

Savić, Miroslav M.; Clayton, Terry; Furtmüller, Roman; Gavrilović, Ivana; Samardžić, Janko; Savić, Snežana; Huck, Sigismund; Sieghart, Werner; Cook, James M.

2008-01-01

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABAA receptors containing α1, α2, α3 or α5 subunits, and may have numerous experimental and clinical applications. The ability of nonselective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the α1 and/or α5 subunit-containing GABAA receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesised and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at α5-containing GABAA receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2’F) selective for GABAA receptors containing the α5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist β-CCt exhibiting a preferential affinity for α1-subunit containing receptors. These data suggest that moderate negative modulation at GABAA receptors containing the α5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined. PMID:18394590

Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds.

PubMed

Shirai, Junya; Tomata, Yoshihide; Kono, Mitsunori; Ochida, Atsuko; Fukase, Yoshiyuki; Sato, Ayumu; Masada, Shinichi; Kawamoto, Tetsuji; Yonemori, Kazuko; Koyama, Ryoukichi; Nakagawa, Hideyuki; Nakayama, Masaharu; Uga, Keiko; Shibata, Akira; Koga, Keiko; Okui, Toshitake; Shirasaki, Mikio; Skene, Robert; Sang, BiChing; Hoffman, Isaac; Lane, Wes; Fujitani, Yasushi; Yamasaki, Masashi; Yamamoto, Satoshi

2018-01-15

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human uterine and placental arteries exhibit tissue-specific acute responses to 17β-estradiol and estrogen-receptor-specific agonists.

PubMed

Corcoran, Jemma J; Nicholson, Christopher; Sweeney, Michèle; Charnock, Jayne C; Robson, Stephen C; Westwood, Melissa; Taggart, Michael J

2014-05-01

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17β-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17β-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10(-6) M), and then incubated with incremental doses (5 min, 0.03-30 µM) of either 17β-estradiol or agonists specific for the ERs ERα (PPT), ERβ (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERβ mRNA expression was assessed. 17β-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17β-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERβ was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17β-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Synthesis and serotonergic activity of 3-[2-(pyrrolidin-1-yl)ethyl]indoles: potent agonists for the h5-HT1D receptor with high selectivity over the h5-HT1B receptor.

PubMed

Sternfeld, F; Guiblin, A R; Jelley, R A; Matassa, V G; Reeve, A J; Hunt, P A; Beer, M S; Heald, A; Stanton, J A; Sohal, B; Watt, A P; Street, L J

1999-02-25

The design, synthesis, and biological evaluation of a novel series of 3-[2-(pyrrolidin-1-yl)ethyl]indoles with excellent selectivity for h5-HT1D (formerly 5-HT1Dalpha) receptors over h5-HT1B (formerly 5-HT1Dbeta) receptors are described. Clinically effective antimigraine drugs such as Sumatriptan show little selectivity between h5-HT1D and h5-HT1B receptors. The differential expression of h5-HT1D and h5-HT1B receptors in neural and vascular tissue prompted an investigation of whether a compound selective for the h5-HT1D subtype would have the same clinical efficacy but with reduced side effects. The pyrrolidine 3b was initially identified as having 9-fold selectivity for h5-HT1D over h5-HT1B receptors. Substitution of the pyrrolidine ring of 3b with methylbenzylamine groups gave compounds with nanomolar affinity for the h5-HT1D receptor and 100-fold selectivity with respect to h5-HT1B receptors. Modification of the indole 5-substituent led to the oxazolidinones 24a,b with up to 163-fold selectivity for the h5-HT1D subtype and improved selectivity over other serotonin receptors. The compounds were shown to be full agonists by measurement of agonist-induced [35S]GTPgammaS binding in CHO cells expressed with h5-HT receptors. This study suggests that the h5-HT1D and h5-HT1B receptors can be differentiated by appropriate substitution of the ligand in the region which binds to the aspartate residue and reveals a large binding pocket in the h5-HT1D receptor domain which is absent for the h5-HT1B receptor. The compounds described herein will be important tools to delineate the role of h5-HT1D receptors in migraine.

Emerging drugs for neuropathic pain.

PubMed

Gilron, Ian; Dickenson, Anthony H

2014-09-01

Neuropathic pain is a costly and disabling condition, which affects up to 8% of the population. Available therapies often provide incomplete pain relief and treatment-related side effects are common. Preclinical neuropathic pain models have facilitated identification of several promising targets, which have progressed to human clinical phases of evaluation. A systematic database search yielded 25 new molecular entities with specified pharmacological mechanisms that have reached Phase II or III clinical trials. These include calcium channel antagonists, vanilloid receptor antagonists, potassium channel agonists, NMDA antagonists, novel opioid receptor agonists, histamine H3 receptor antagonists, a novel sodium channel antagonist, serotonin modulators, a novel acetylcholine receptor agonist, α-2b adrenoreceptor agonist, cannabinoid CB2 receptor agonist, nitric oxide synthase inhibitor, orexin receptor antagonist, angiotensin II 2 antagonist, imidazoline I2 receptor agonist, apoptosis inhibitor and fatty acid amide hydrolase inhibitor. Although the diversity of pharmacological mechanisms of interest emphasise the complexity of neuropathic pain transmission, the considerable number of agents under development reflect a continued enthusiasm in drug development for neuropathic pain. Ongoing enhancements in methodology of both preclinical and clinical research and closer translation in both directions are expected to more efficiently identify new agents, which will improve the management of neuropathic pain.

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands*

PubMed Central

Kapur, Ankur; Zhao, Pingwei; Sharir, Haleli; Bai, Yushi; Caron, Marc G.; Barak, Larry S.; Abood, Mary E.

2009-01-01

The cannabinoid receptor 1 (CB1) and CB2 cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy. PMID:19723626

NMDA receptor adjusted co-administration of ecstasy and cannabinoid receptor-1 agonist in the amygdala via stimulation of BDNF/Trk-B/CREB pathway in adult male rats.

PubMed

Ashabi, Ghorbangol; Sadat-Shirazi, Mitra-Sadat; Khalifeh, Solmaz; Elhampour, Laleh; Zarrindast, Mohammad-Reza

2017-04-01

Consumption of cannabinoid receptor-1 (CB-1) agonist such as cannabis is widely taken in 3,4- methylenedioxymethamphetamine (MDMA) or ecstasy users; it has been hypothesized that co-consumption of CB-1 agonist might protect neurons against MDMA toxicity. N-methyl-d-aspartate (NMDA) receptors regulate neuronal plasticity and firing rate in the brain through Tyrosine-kinase B (Trk-B) activation. The molecular and electrophysiological association among NMDA and MDMA/Arachidonylcyclopropylamide (ACPA, a selective CB-1 receptor agonist) co-consumption was not well-known. Here, neuronal spontaneous activity, Brain-derived neurotrophic factor (BDNF), Trk-B and cAMP response element binding protein (CREB) phosphorylation levels were recognized in ACPA and MDMA co-injected rats. Besides, we proved the role of NMDA receptor on MDMA and ACPA combination on neuronal spontaneous activity and Trk-B/BDNF pathway in the central amygdala (CeA). Male rats were anesthetized with intra-peritoneal injections of urethane; MDMA, D-2-amino-5-phosphonopentanoate (D-AP5, NMDA receptor antagonist) were injected into CeA. ACPA was administrated by intra-cerebroventricular injection. Thirty minutes following injections, neuronal firing rate was recorded from CeA. Two hours after drug injection, amygdala was collected from brain for molecular evaluations. Single administration of MDMA and/or ACPA reduced firing rates compared with sham group in the CeA dose-dependently. Injection of D-AP5, ACPA and MDMA reduced firing rate compared with sham group (P<0.001). Interestingly, injection of ACPA+MDMA enhanced BDNF, Trk-B and CREB phosphorylation compared with MDMA groups. D-AP5, ACPA and MDMA co-injection decreased BDNF, Trk-B and CREB phosphorylation levels compared with ACPA+MDMA in the amygdala (P<0.01). Probably, NMDA receptors are involved in the protective role of acute MDMA+ACPA co-injection via BDNF/Trk-B/CREB pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia

PubMed Central

Bristow, Linda J.; Easton, Amy E.; Li, Yu-Wen; Sivarao, Digavalli V.; Lidge, Regina; Jones, Kelli M.; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J.; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E.; Zaczek, Robert; Olson, Richard

2016-01-01

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID:27467081

Differential contributions of serotonin receptors to the behavioral effects of indoleamine hallucinogens in mice

PubMed Central

Halberstadt, Adam L; Koedood, Liselore; Powell, Susan B; Geyer, Mark A

2012-01-01

Psilocin (4-hydroxy-N,N-dimethyltryptamine) is a hallucinogen that acts as an agonist at 5-HT1A, 5-HT2A, and 5-HT2C receptors. Psilocin is the active metabolite of psilocybin, a hallucinogen that is currently being investigated clinically as a potential therapeutic agent. In the present investigation, we used a combination of genetic and pharmacological approaches to identify the serotonin (5-HT) receptor subtypes responsible for mediating the effects of psilocin on head twitch response (HTR) and the behavioral pattern monitor (BPM) in C57BL/6J mice. We also compared the effects of psilocin with those of the putative 5-HT2C receptor-selective agonist 1-methylpsilocin and the hallucinogen and non-selective serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT). Psilocin, 1-methylpsilocin, and 5-MeO-DMT induced the HTR, effects that were absent in mice lacking the 5-HT2A receptor gene. When tested in the BPM, psilocin decreased locomotor activity, holepoking, and time spent in the center of the chamber, effects that were blocked by the selective 5-HT1A antagonist WAY-100635 but were not altered by the selective 5-HT2C antagonist SB 242,084 or by 5-HT2A receptor gene deletion. 5-MeO-DMT produced similar effects when tested in the BPM, and the action of 5-MeO-DMT was significantly attenuated by WAY-100635. Psilocin and 5-MeO-DMT also decreased the linearity of locomotor paths, effects that were mediated by 5-HT2C and 5-HT1A receptors, respectively. In contrast to psilocin and 5-MeO-DMT, 1-methylpsilocin (0.6–9.6 mg/kg) was completely inactive in the BPM. These findings confirm that psilocin acts as an agonist at 5-HT1A, 5-HT2A, and 5-HT2C receptors in mice, whereas the behavioral effects of 1-methylpsilocin indicate that this compound is acting at 5-HT2A sites but is inactive at the 5-HT1A receptor. The fact that 1-methylpsilocin displays greater pharmacological selectivity than psilocin indicates that 1-methylpsilocin represents a potentially useful alternative to psilocybin for development as a potential therapeutic agent. PMID:21148021

Peroxisome proliferator-activated receptor δ agonists inhibit T helper type 1 (Th1) and Th17 responses in experimental allergic encephalomyelitis

PubMed Central

Kanakasabai, Saravanan; Chearwae, Wanida; Walline, Crystal C; Iams, Wade; Adams, Suzanne M; Bright, John J

2010-01-01

Multiple sclerosis (MS) is a neurological disorder that affects more than a million people world-wide. The aetiology of MS is not known and there is no medical treatment available that can cure MS. Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune disease model of MS. The pathogenesis of EAE/MS is a complex process involving activation of immune cells, secretion of inflammatory cytokines and destruction of myelin sheath in the central nervous system (CNS). Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptor transcription factors that regulate cell growth, differentiation and homeostasis. PPAR agonists have been used in the treatment of obesity, diabetes, cancer and inflammation. We and others have shown that PPARγ, α and δ agonists inhibit CNS inflammation and demyelination in the EAE model of MS. In this study we show that the PPARδ agonists GW501516 and L165041 ameliorate MOGp35-55-induced EAE in C57BL/6 mice by blocking interferon (IFN)-γ and interleukin (IL)-17 production by T helper type 1 (Th1) and Th17 cells. The inhibition of EAE by PPARδ agonists was also associated with a decrease in IL-12 and IL-23 and an increase in IL-4 and IL-10 expression in the CNS and lymphoid organs. These findings indicate that PPARδ agonists modulate Th1 and Th17 responses in EAE and suggest their use in the treatment of MS and other autoimmune diseases. PMID:20406305

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB(1) receptor antagonist for the management of obesity.

PubMed

Hadcock, John R; Griffith, David A; Iredale, Phillip A; Carpino, Phillip A; Dow, Robert L; Black, Shawn C; O'Connor, Rebecca; Gautreau, Denise; Lizano, Jeffrey S; Ward, Karen; Hargrove, Diane M; Kelly-Sullivan, Dawn; Scott, Dennis O

2010-04-02

Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system. 2010 Elsevier Inc. All rights reserved.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

PubMed Central

McNamara, Fergal N; Randall, Andrew; Gunthorpe, Martin J

2005-01-01

We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. The current–voltage relationship of piperine-activated currents showed pronounced outward rectification (25±4-fold between −70 and +70 mV) and a reversal potential of 0.0±0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. Although piperine was a less potent agonist (EC50=37.9±1.9 μM) than capsaicin (EC50=0.29±0.05 μM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230±20%, n=11) or capsaicin (284±32%, n=8) upon acidification to pH 6.5. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t1/2=9.9±0.7 s) than capsaicin (t1/2>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function. PMID:15685214

Peroxisome proliferator-activated receptor agonists modulate neuropathic pain: a link to chemokines?

PubMed Central

Freitag, Caroline M.; Miller, Richard J.

2014-01-01

Chronic pain presents a widespread and intractable medical problem. While numerous pharmaceuticals are used to treat chronic pain, drugs that are safe for extended use and highly effective at treating the most severe pain do not yet exist. Chronic pain resulting from nervous system injury (neuropathic pain) is common in conditions ranging from multiple sclerosis to HIV-1 infection to type II diabetes. Inflammation caused by neuropathy is believed to contribute to the generation and maintenance of neuropathic pain. Chemokines are key inflammatory mediators, several of which (MCP-1, RANTES, MIP-1α, fractalkine, SDF-1 among others) have been linked to chronic, neuropathic pain in both human conditions and animal models. The important roles chemokines play in inflammation and pain make them an attractive therapeutic target. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors known for their roles in metabolism. Recent research has revealed that PPARs also play a role in inflammatory gene repression. PPAR agonists have wide-ranging effects including inhibition of chemokine expression and pain behavior reduction in animal models. Experimental evidence suggests a connection between the pain ameliorating effects of PPAR agonists and suppression of inflammatory gene expression, including chemokines. In early clinical research, one PPARα agonist, palmitoylethanolamide (PEA), shows promise in relieving chronic pain. If this link can be better established, PPAR agonists may represent a new drug therapy for neuropathic pain. PMID:25191225

Pharmacological characterization of human recombinant melatonin mt1 and MT2 receptors

PubMed Central

Browning, Christopher; Beresford, Isabel; Fraser, Neil; Giles, Heather

2000-01-01

We have pharmacologically characterized recombinant human mt1 and MT2 receptors, stably expressed in Chinese hamster ovary cells (CHO-mt1 and CHO-MT2), by measurement of [3H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt1 and MT2 receptors with pKD values of 9.89 and 9.56 and Bmax values of 1.20 and 0.82 pmol mg−1 protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt1 and MT2 receptors, a number of putative antagonists had substantially higher affinities for MT2 receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt1 and CHO-MT2 cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC50 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT2 receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt1 receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt1 and CHO-MT2 cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA2 values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of Gi/Go G-proteins. This is the first report of the use of [3H]-melatonin for the characterization of recombinant mt1 and MT2 receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles. PMID:10696085

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

The role of serotonin-2 (5-HT2) and dopamine receptors in the behavioral actions of the 5-HT2A/2C agonist, DOI, and putative 5-HT2C inverse agonist, SR46349B.

PubMed

Scarlota, Laura C; Harvey, John A; Aloyo, Vincent J

2011-02-01

Atypical antipsychotic efficacy is often attributed to actions at serotonin-2 (5-HT(2)) and dopamine receptors, indicating a potential benefit of understanding the interplay between these systems. Currently, it is known that 5-HT(2) receptors modulate dopamine release, although the role of specific dopamine receptors in 5-HT(2)-mediated behavior is not well understood. We examined the role of 5-HT(2A), 5-HT(2C), and dopamine (D1 and D2) receptors in the behavioral response to a 5-HT(2A/2C) agonist (DOI) and 5-HT(2A/2C) antagonist (SR46349B). Effects were assessed by measuring rabbit head bobs (previously characterized as 5-HT(2A) receptor-mediated) and body shakes (5-HT(2C)-mediated). As expected, DOI produced head bobs and body shakes, and these DOI-elicited behaviors were attenuated by the SR46349B pretreatment. Unexpectedly, SR46349B also induced head bobs when administered alone. However, SR46349B-elicited head bobs are distinguishable from those produced by DOI since the 5-HT(2A) antagonist, ketanserin, only attenuated DOI-elicited head bobs. Conversely, 5-HT(2C) ligands (SB242084 and SB206553) inhibited SR46349B but not DOI-induced head bobs. Furthermore, when administered alone, SB206553 (a 5-HT(2C) inverse agonist) produced head bobs, indicating the behavior can be either 5-HT(2A) or 5-HT(2C) mediated. Next, it was revealed that D1 and D2 receptors play a role in DOI-elicited head bobs, but only D1 receptors are required for SR46349B-elicited head bobs. 5-HT(2A) receptor agonism and 5-HT(2C) inverse agonism produce the same behavior, likely due to similar downstream actions at D1 receptors. Consequently, 5-HT(2C) agonism or D1 agonism may be effective therapies for disorders, such as schizophrenia, currently being treated with 5-HT(2A) antagonists.

Modified melanocortin tetrapeptide Ac-His-dPhe-Arg-Trp-NH at the arginine side chain with ureas and thioureas.

PubMed

Joseph, C G; Sorensen, N B; Wood, M S; Xiang, Z; Moore, M C; Haskell-Luevano, C

2005-11-01

The Ac-His-dPhe-Arg-Trp-NH2 tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3-5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea- and thiourea-substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3-5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3-5 receptors. The Arg side chain-modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side-chain template. These ligands elicit full-agonist pharmacology at the mouse MCRs examined in this study.

Thinking in cycles: MWC is a good model for acetylcholine receptor-channels

PubMed Central

Auerbach, Anthony

2012-01-01

Abstract Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ∼–5.1 kcal mol−1 towards the global gating isomerization of the protein. PMID:21807612

Mixed Kappa/Mu Opioid Receptor Agonists: The 6β-Naltrexamines

PubMed Central

Cami-Kobeci, Gerta; Neal, Adrian P.; Bradbury, Faye A.; Purington, Lauren C.; Aceto, Mario D.; Harris, Louis S.; Lewis, John W.; Traynor, John R.; Husbands, Stephen M.

2011-01-01

Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6β-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, non-selective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist, and therefore somewhat similar to the previously evaluated analogues 3–6, while 12b displayed predominant MOR agonist activity. PMID:19253970

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

PubMed

Maggi, C A; Patacchini, R; Eglezos, A; Quartara, L; Giuliani, S; Giachetti, A

1992-09-01

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged. 6. The selective NK2 receptor antagonists MEN 10,376, L 659,877 and R 396 antagonized competitively the response to [PAla8] neurokinin A (4-10) with the following rank order of potency (pA2 values in parentheses): MEN 10,376 (7.41)>L 659,877 (7.15)>R 396 (6.43). MEN 10,376 and L 659,877 also competitively antagonized the response to neurokinin A, although with lower potency as compared to the selective NK2 receptor agonist.7. MEN 10,376, L 659,877 and R 396 reduced in a concentration-dependent manner the contractile response produced by electrical field stimulation (1 Hz, 100 V, 0.25 ms pulse width, trains of 10 s). The rank order of potency of NK2 receptor antagonists in blocking the response to electrical stimulation (MEN 10,376> L 659,877> R 396) closely mimicked their potency in antagonizing exogenous tachykinins.8. The inhibitory effect of MEN 10,376 toward responses produced by electrical field stimulation was significantly reduced when tested in the presence of peptidase inhibitors, which increased significantly the response to nerve stimulation.9. GR 82,334 (3 pM) did not significantly affect the response to nerve stimulation in untreated preparations and slightly reduced it in the presence of peptidase inhibitors.10. We conclude that both NK, and NK2 receptors mediate the contractile effect of tachykinins in the circular muscle of the guinea-pig renal pelvis and that the response ascribable to NK2 receptor stimulation is larger than that ascribed to NK, receptor stimulation. The NK2 receptor in the guinea-pig renal pelvis belongs to the same subtype previously identified in the rabbit pulmonary artery. NK2 receptors play a dominant role in the physiological response determined by the release of endogenous tachykinins and a contribution of NKI receptors becomes evident after inhibition of peptide degradation.

Role of hypothalamic cannabinoid receptors in post-stroke depression in rats.

PubMed

Wang, Shanshan; Sun, Hong; Liu, Sainan; Wang, Ting; Guan, Jinqun; Jia, Jianjun

2016-03-01

One of the most common psychological consequences of stroke is post-stroke depression (PSD). While more than 30 percent of stroke patients eventually develop PSD, the neurobiological mechanisms underlying such a phenomenon have not been well investigated. Given the critical involvement of hypothalamic-pituitary-adrenal axis and endocannabinoid system in response to stressful stimuli, we evaluated the hypothesis that cannabinoid receptors in the hypothalamus are critical for modulation of post-stroke depression-like behaviors in rats. To this end, rats were treated with middle cerebral artery occlusion (MCAO) followed by chronic unpredictable mild stress (CUMS) treatment procedure. We then assessed the expression of CB1 and CB2 receptors in the hypothalamus, and evaluated the effects of pharmacological stimulations of CB1 or CB2 receptors on the expression and development of depression-like behaviors in PSD rats. We found that PSD rats exhibited decreased the expression of CB1 receptor, but not CB2 receptor, in the ventral medial hypothalamus (VMH). Such an effect was not observed in the dorsally adjacent brain regions. Furthermore, intra-VMH injections of CB2 receptor agonist, but not CB1 receptor agonist, attenuated the expression of depression-like behaviors in PSD rats. Finally, repeated intraperitoneal injections of CB1 or CB2 receptor agonists during CUMS treatment inhibited the development of depression-like behaviors in PSD rats. Taken together, these results suggest that decreased CB1 receptor expression is likely associated with the development of post-stroke depression, and CB2 receptor may be a potential therapeutic target for the treatment post-stroke depressive disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Agonism and Antagonism at the Insulin Receptor

PubMed Central

Knudsen, Louise; Hansen, Bo Falck; Jensen, Pia; Pedersen, Thomas Åskov; Vestergaard, Kirsten; Schäffer, Lauge; Blagoev, Blagoy; Oleksiewicz, Martin B.; Kiselyov, Vladislav V.; De Meyts, Pierre

2012-01-01

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed. PMID:23300584

Ipragliflozin Add-on Therapy to a GLP-1 Receptor Agonist in Japanese Patients with Type 2 Diabetes (AGATE): A 52-Week Open-Label Study.

PubMed

Ishihara, Hisamitsu; Yamaguchi, Susumu; Nakao, Ikko; Sakatani, Taishi

2018-06-20

Few data are available regarding ipragliflozin treatment in combination with glucagon-like peptide-1 (GLP-1) receptor agonists. The aim of this study was to evaluate the efficacy and safety of ipragliflozin in combination with GLP-1 receptor agonists in Japanese patients with inadequately controlled type 2 diabetes mellitus (T2DM). This multicenter study (consisting of three periods: a 4-week washout period, a 6-week observation period, and a 52-week open-label treatment period) included patients aged ≥ 20 years who received a stable dose/regimen of a GLP-1 receptor agonist either solely or in combination therapy with a sulfonylurea for ≥ 6 weeks, with glycosylated hemoglobin (HbA1c) of ≥ 7.5% and a fasting plasma glucose (FPG) of ≥ 126 mg/dL. Ipragliflozin treatment was given at a fixed dose of 50 mg/day for 20 weeks, followed by 50 or 100 mg/day for 32 weeks. Changes from baseline in glycemic control and other parameters were examined; safety was also assessed. The mean changes in HbA1c and body weight from baseline to end of treatment were - 0.92% and - 2.69 kg, respectively, in all ipragliflozin-treated patients (n = 103). Overall, sustained reductions from baseline were observed for HbA1c, FPG, self-monitored blood glucose, and body weight during the 52-week treatment. The dose increase of ipragliflozin to 100 mg/day resulted in better glycemic control and weight reduction for patients in whom the 50-mg dose was insufficient. Overall, 46.6% (48/103) of patients experienced drug-related adverse events. The most common drug-related treatment-emergent adverse events were pollakiuria (9.7%), hypoglycemia (8.7%), constipation (6.8%), and thirst (5.8%). Combined therapy with ipragliflozin and GLP-1 receptor agonists/sulfonylureas was significantly efficacious in reducing glycemic parameters in patients with T2DM with inadequate glycemic control, and no major safety concerns were identified. The results from this study suggest that ipragliflozin can be recommended as a well-tolerated and effective add-on therapy to a GLP-1 receptor agonist for the treatment of T2DM. ClinicalTrials.gov (identifier: NCT02291874). Astellas Pharma Inc., Tokyo, Japan.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

PubMed

Kurt, Akif Hakan; Buyukafsar, Kansu

2013-02-28

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17β-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney. Copyright © 2013 Elsevier B.V. All rights reserved.

Affinity purification of angiotensin type 2 receptors from N1E-115 cells: evidence for agonist-induced formation of multimeric complexes.

PubMed

Siemens, I R; Yee, D K; Reagan, L P; Fluharty, S J

1994-01-01

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

PubMed

Vecchio, Elizabeth A; Chuo, Chung Hui; Baltos, Jo-Anne; Ford, Leigh; Scammells, Peter J; Wang, Bing H; Christopoulos, Arthur; White, Paul J; May, Lauren T

2016-10-01

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of GABA receptor agonists or antagonists injected spinally on the blood glucose level in mice.

PubMed

Sim, Yun-Beom; Park, Soo-Hyun; Kang, Yu-Jung; Kim, Sung-Su; Kim, Chea-Ha; Kim, Su-Jin; Jung, Jun-Sub; Ryu, Ohk-Hyun; Choi, Moon-Gi; Suh, Hong-Won

2013-05-01

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABAB receptor agonist; 1-10 μg/5 μl) or bicuculline (a GABAA receptor antagonist; 1-10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABAA receptor agonist; 1-5 μg/5 μl) or phaclofen (a GABAB receptor antagonist; 5-10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen-induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABAB receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABAA receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.

The GABA(B) receptor positive modulator BHF177 attenuated anxiety, but not conditioned fear, in rats.

PubMed

Li, Xia; Kaczanowska, Katarzyna; Finn, M G; Markou, Athina; Risbrough, Victoria B

2015-10-01

GABAB (γ-aminobutyric acid B) receptors may be a therapeutic target for anxiety disorders. Here we characterized the effects of the GABAB receptor positive allosteric modulator (PAM) BHF177 on conditioned and unconditioned physiological responses to threat in the light-enhanced startle (LES), stress-induced hyperthermia, and fear-potentiated startle (FPS) procedures in rats. The effects of BHF177 on LES were compared with those of the GABAB receptor agonists baclofen and CGP44532, and the positive control buspirone, a 5-HT1A receptor partial agonist with anxiolytic activity in humans. Baclofen (0.4, 0.9 and 1.25 mg/kg) and CGP44532 (0.065, 0.125 and 0.25 mg/kg) administration had significant sedative, but not anxiolytic, activity reflected in overall decrease in the startle response in the LES tests. BHF177 (10, 20 and 40 mg/kg) had no effect on LES, nor did it produce an overall sedative effect. Interesting, however, when rats were grouped by high and low LES responses, BHF177 had anxiolytic-like effects only on LES in high, but not low, LES responding rats. BHF177 also blocked stress-induced hyperthermia, but had no effect on conditioned fear responses in the FPS test. Buspirone (1 and 3 mg/kg) had an anxiolytic-like profile in both LES and FPS tests. These results indicate that BHF177 may specifically attenuate unconditioned anxiety in individuals that exhibit a high anxiety state, and has fewer sedative effects than direct agonists. Thus, BHF177 or other GABAB receptor PAMs may be promising compounds for alleviating increased anxiety seen in various psychiatric disorders with a superior side-effect profile compared to GABAB receptor agonists. Published by Elsevier Ltd.

Mu/Kappa Opioid Interactions in Rhesus Monkeys: Implications for Analgesia and Abuse Liability

PubMed Central

Negus, S. Stevens; Katrina Schrode, KA; Stevenson, Glenn W.

2008-01-01

Mu opioid receptor agonists are clinically valuable as analgesics; however, their use is limited by high abuse liability. Kappa opioid agonists also produce antinociception, but they do not produce mu agonist-like abuse-related effects, suggesting that they may enhance the antinociceptive effects and/or attenuate the abuse-related effects of mu agonists. To evaluate this hypothesis, the present study examined interactions between the mu agonist fentanyl and the kappa agonist U69,593 in three behavioral assays in rhesus monkeys. In an assay of schedule-controlled responding, monkeys responded under a fixed-ratio 30 (FR 30) schedule of food presentation. Fentanyl and U69,593 each produced rate-decreasing effects when administered alone, and mixtures of 0.22:1, 0.65:1 and 1.96:1 U69,593/fentanyl usually produced subadditive effects. In an assay of thermal nociception, tail withdrawal latencies were measured from water heated to 50°C. Fentanyl and U69,593 each produced dose-dependent antinociception, and effects were additive for all mixtures. In an assay of drug self-administration, rhesus monkeys responded for i.v. drug injection, and both dose and FR values were manipulated. Fentanyl maintained self-administration, whereas U69,593 did not. Addition of U69,593 to fentanyl produced a proportion-dependent decrease in both rates of fentanyl self-administration and behavioral economic measures of the reinforcing efficacy of fentanyl. Taken together, these results suggest that simultaneous activation of mu and kappa receptors, either with a mixture of selective drugs or with a single drug that targets both receptors, may reduce abuse liability without reducing analgesic effects relative to selective mu agonists administered alone. PMID:18837635

A Novel Aminotetralin-Type Serotonin (5-HT) 2C Receptor-Specific Agonist and 5-HT2A Competitive Antagonist/5-HT2B Inverse Agonist with Preclinical Efficacy for Psychoses

PubMed Central

Morgan, Drake; Felsing, Daniel; Kondabolu, Krishnakanth; Rowland, Neil E.; Robertson, Kimberly L.; Sakhuja, Rajeev; Booth, Raymond G.

2014-01-01

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 Pesticides with Different Modes of Action

DTIC Science & Technology

2008-01-01

imidacloprid , spinosad, diazinon, and carbaryl showed slightly lower activity than permethrin (20-fold). However, bifenazate showed very low activity...acetylcholinesterase inhibitor representing organophosphates (LD50 6.7 10 4 g/mg) imidacloprid , a nicotinic acetylcholine re- ceptor...inhibitors (Carbamates) 1A Spinosad Nicotinic acetylcholine receptor agonists 5 Imidacloprid Nicotinic acetylcholine receptor agonist/antagonists 4

Analytical confirmation of synthetic cannabinoids in a cohort of 179 presentations with acute recreational drug toxicity to an Emergency Department in London, UK in the first half of 2015.

PubMed

Abouchedid, Rachelle; Hudson, Simon; Thurtle, Natalie; Yamamoto, Takahiro; Ho, James H; Bailey, George; Wood, Michelle; Sadones, Nele; Stove, Christophe P; Dines, Alison; Archer, John R H; Wood, David M; Dargan, Paul I

2017-06-01

Synthetic cannabinoid receptor agonists are the largest group of new psychoactive substances reported in the last decade; in this study we investigated how commonly these drugs are found in patients presenting to the Emergency Department with acute recreational drug toxicity. We conducted an observational cohort study enrolling consecutive adult patients presenting to an Emergency Department (ED) in London (UK) January-July 2015 (6 months) with acute recreational drug toxicity. Residual serum obtained from a serum sample taken as part of routine clinical care was analyzed using high-resolution accurate mass-spectrometry with liquid-chromatography (HRAM-LCMSMS). Minimum clinical data were obtained from ED medical records. 18 (10%) of the 179 patient samples were positive for synthetic cannabinoid receptor agonists. The most common was 5F AKB-48 (13 samples, concentration 50-7600 pg/ml), followed by 5F PB-22 (7, 30-400 pg/mL), MDMB-CHMICA (7, 80-8000 pg/mL), AB-CHMINACA (3, 50-1800 pg/mL), Cumyl 5F-PINACA (1, 800 pg/mL) and BB-22 (1, 60 pg/mL). Only 9/18 (50%) in whom synthetic cannabinoid receptor agonists were detected self-reported synthetic cannabinoid receptor agonist use. The most common clinical features were seizures and agitation, both recorded in four (22%) individuals. Fourteen patients (78%) were discharged from the ED, one of the four admitted to hospital was admitted to critical care. Synthetic cannabinoid receptor agonists were found in 10% of this cohort with acute recreational drug toxicity but self-reported in only half of these. This suggests that presentations to the ED with acute synthetic cannabinoid receptor agonist toxicity may be more common than reported.

Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer.

PubMed

Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Ferré, Sergi

2014-10-01

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. U.S. Government work not protected by U.S. copyright.

Stereochemical requirements for central and peripheral muscarinic and antimuscarinic activity of some acetylenic compounds related to oxotremorine.

PubMed Central

Dahlbom, R.; Jenden, D. J.; Resul, B.; Ringdahl, B.

1982-01-01

1 The enantiomers of some analogues of the central muscarinic agent, oxotremorine, were prepared and investigated for tremorogenic and tremorolytic activity in intact mice and for muscarinic and antimuscarinic activity on the isolated ileum of the guinea-pig. 2 The R-isomers were more potent than the S-isomers both in vivo and in vitro regardless of whether the compounds are agonists, partial agonists or competitive antagonists. 3 It is suggested that in the oxotremorine series, agonists and antagonists interact with a common receptor site, in contrast to classical muscarinic antagonists which are believed to bind also to accessory receptor areas, located close to the agonist binding site. PMID:7093587

Selective central activation of somatostatin receptor 2 increases food intake, grooming behavior and rectal temperature in rats.

PubMed

Stengel, A; Goebel, M; Wang, L; Rivier, J; Kobelt, P; Monnikes, H; Tache, Y

2010-08-01

The consequences of selective activation of brain somatostatin receptor-2 (sst2) were assessed using the sst2 agonist, des-AA(1,4-6,11-13)-[DPhe(2),Aph7(Cbm),DTrp(8)]-Cbm-SST-Thr-NH2. Food intake (FI) was monitored in ad libitum fed rats chronically implanted with an intracerebroventricular (i.c.v.) cannula. The sst(2) agonist injected i.c.v. at 0.1 and 1 microg/rat dose-dependently increased light phase FI from 2 to 6 hours post injection (2.3+/-0.5 and 7.5+/-1.2 respectively vs. vehicle: 0.2+/-0.2 g/300 g bw, P<0.001). Peptide action was reversed by i.c.v. injection of the sst2 antagonist, des-AA(1,4-6,11-13)-[pNO(2)-Phe(2),DCys(3),Tyr(7),DAph(Cbm)8]-SST-2Nal-NH(2) and not reproduced by intraperitoneal injection (30 microg/rat). The sst(2) antagonist alone i.c.v. significantly decreased the cumulative 14-hours dark phase FI by 29.5%. Other behaviors, namely grooming, drinking and locomotor activity were also increased by the sst(2) agonist (1 microg/rat, i.c.v.) as monitored during the 2(nd) hour post injection while gastric emptying of solid food was unaltered. Rectal temperature rose 1 hour after the sst(2) agonist (1 microg/rat, i.c.v.) with a maximal response maintained from 1 to 4 hours post injection. These data show that selective activation of the brain sst(2) receptor induces a feeding response in the light phase not associated with changes in gastric emptying. The food intake reduction following sst(2) receptor blockade suggests a role of this receptor in the orexigenic drive during the dark phase.

Pharmacological profiles of cloned mammalian P2Y-receptor subtypes.

PubMed

von Kügelgen, Ivar

2006-06-01

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and functionally defined; species orthologues have been found in many vertebrates. P2Y1-, P2Y2-, P2Y4-, P2Y6-, and P2Y11-receptors all couple to stimulation of phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase. In contrast, activation of the P2Y12-, P2Y13-, and P2Y14-receptors causes an inhibition of adenylate cyclase activity. The expression of P2Y1-receptors is widespread. The receptor is involved in blood platelet aggregation, vasodilatation and neuromodulation. It is activated by ADP and ADP analogues including 2-methylthio-ADP (2-MeSADP). 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS2179) and 2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2279) are potent and selective antagonists. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y2-receptor is activated by UTP and ATP and blocked by suramin. The P2Y2-agonist diquafosol is used for the treatment of the dry eye disease. P2Y4-receptors are expressed in the placenta and in epithelia. The human P2Y4-receptor has a strong preference for UTP as agonist, whereas the rat P2Y4-receptor is activated about equally by UTP and ATP. The P2Y4-receptor is not blocked by suramin. The P2Y6-receptor has a widespread distribution including heart, blood vessels, and brain. The receptor prefers UDP as agonist and is selectively blocked by 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567). The P2Y11-receptor may play a role in the differentiation of immunocytes. The human P2Y11-receptor is activated by ATP as naturally occurring agonist and it is blocked by suramin and reactive blue 2 (RB2). The P2Y12-receptor plays a crucial role in platelet aggregation as well as in inhibition of neuronal cells. It is activated by ADP and very potently by 2-methylthio-ADP. Nucleotide antagonists including N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (=cangrelor; AR-C69931MX), the nucleoside analogue AZD6140, as well as active metabolites of the thienopyridine compounds clopidogrel and prasugrel block the receptor. These P2Y12-antagonists are used in pharmacotherapy to inhibit platelet aggregation. The P2Y13-receptor is expressed in immunocytes and neuronal cells and is again activated by ADP and 2-methylthio-ADP. The 2-chloro-5-nitro pyridoxal-phosphate analogue 6-(2'-chloro-5'-nitro-azophenyl)-pyridoxal-alpha5-phosphate (MRS2211) is a selective antagonist. mRNA encoding for the human P2Y14-receptor is found in many tissues. However, a physiological role of the receptor has not yet been established. UDP-glucose and related analogues act as agonists; antagonists are not known. Finally, UDP has been reported to act on receptors for cysteinyl leukotrienes as an additional agonist--indicating a dual agonist specificity of these receptors.

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

PubMed Central

Friesen, Claudia; Hormann, Inis; Roscher, Mareike; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf; Debatin, Klaus-Michael; Miltner, Erich

2014-01-01

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells. PMID:24626197

Sensitivity of bronchopulmonary receptors to cold and heat mediated by transient receptor potential cation channel subtypes in an ex vivo rat lung preparation.

PubMed

Zhou, Yun; Sun, Biying; Li, Qian; Luo, Pin; Dong, Li; Rong, Weifang

2011-08-15

Changes in airway temperature can result in respiratory responses such as cough, bronchoconstriction and mucosal secretion after cold exposure and hyperventilation after heat exposure. In the present investigation, we examined the activity of bronchopulmonary receptors in response to activators of thermo-sensitive transient receptor potential (TS-TRP) cation channels using an ex vivo rat lung preparation. Receptive fields in small bronchioles were probed with von Frey hair monofilaments, warm (50°C) or cold (8°C) saline or saline containing TS-TRP agonists. Among 233 fibers tested, 159 (68.2%) responded to heat (50°C). A large proportion of heat-responsive receptors (107/145) were also activated by capsaicin. Heat and capsaicin-evoked responses were both blocked by TRPV1 antagonist, capsazepine. Only 15.3% of airway receptors responded to cold, which was associated with sensitivity to TRPM8 agonist menthol but not to TRPA1 agonist cinnamaldehyde (CA). Moreover, cold-evoked responses was unaffected by TRPA1 antagonist HC-03001. Our observations suggest that TRPV1 and TRPM8 are involved in transducing heat and cold in the lower respiratory tract, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

Pharmacological stress myocardial perfusion imaging with the potent and selective A(2A) adenosine receptor agonists ATL193 and ATL146e administered by either intravenous infusion or bolus injection.

PubMed

Glover, D K; Ruiz, M; Takehana, K; Petruzella, F D; Riou, L M; Rieger, J M; Macdonald, T L; Watson, D D; Linden, J; Beller, G A

2001-09-04

Adenosine (Ado) and dipyridamole are alternatives to exercise stress for myocardial perfusion imaging. Though generally safe, side effects frequently occur that cause patient discomfort and sometimes lead to premature termination of the study or require aminophylline administration. Recently, a new class of A(2A) Ado receptor agonists was synthesized. ATL193 and ATL146e are 2-propynylcyclohexyl-5'-N-ethylcarboxamido derivatives of Ado. The study goals were to evaluate the potency and selectivity of these new compounds on recombinant canine Ado receptors and to evaluate their hemodynamic properties in dogs to assess their usefulness as vasodilators for myocardial perfusion imaging. In assays of recombinant canine Ado receptors, ATL-193 and ATL-146e were highly selective for the A(2A) over the A(1) and A(3) receptors and were more potent than MRE-0470 and CGS-21680. In 16 anesthetized dogs, the agonists were administered by infusion (ATL-193; n=7 normal) or bolus injection (ATL-146e; n=9 critical left anterior descending coronary artery stenosis), and hemodynamic responses were compared with those of Ado. Both agonists produced dose-dependent coronary flow (CF) elevation without provoking the hypotension observed with Ado. After an ATL-146e bolus, the CF increase was sustained for several minutes, providing ample time for injection and myocardial uptake of (99m)Tc-sestamibi, and CF returned to baseline within 20 minutes. The CF increase was completely blocked by the selective A(2A) antagonist ZM241385 (3 microgram. kg(-1). min(-1)). ATL-193 and ATL-146e are highly potent and selective Ado A(2A) receptor agonists with excellent potential for use as vasodilators for myocardial perfusion imaging. An important advantage of ATL-146e is the ability to administer it by bolus injection.

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

PubMed Central

Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

2011-01-01

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABAB receptors, but not α2 adrenergic receptors

PubMed Central

Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G.

2010-01-01

GABAB, μ-opioid, and adrenergic α2 receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABAB receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABAB agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α2 adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABAB receptors, but not by α2 receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. PMID:20726886

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

PubMed Central

Bradley, ME; Bond, ME; Manini, J; Brown, Z; Charlton, SJ

2009-01-01

Background and purpose: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. Experimental approach: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [35S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils. Key results: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [125I]-interleukin-8 ([125I]-IL-8) without affecting the Kd. In contrast, IL-8 was unable to prevent binding of [3H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [35S]-GTPγS binding in this preparation. Conclusions and implications: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling. PMID:19422399

Differential effects of R-isovaline and the GABAB agonist, baclofen, in the guinea pig ileum.

PubMed

Fung, Timothy; Asseri, Khalid A; Asiri, Yahya I; Wall, Richard A; Schwarz, Stephan K W; Puil, Ernest; MacLeod, Bernard A

2016-11-15

R-isovaline is a non-proteinogenic amino acid which produces analgesia in a range of nociceptive assays. Mediation of this effect by metabotropic receptors for γ-aminobutyric acid (GABA) and glutamate, demonstrated by previous work, may depend on the type of tissue or receptor system. The objective of this study was to assess the activity of R-isovaline acting at GABA B and group II metabotropic glutamate receptors in guinea pig ileum, which is known to exhibit well-defined responses to GABA B agonists such as baclofen. The effects of bath-applied R-isovaline and RS-baclofen were examined on electrically evoked contractions of guinea pig ileum and during GABA B antagonism by CGP52432. In separate experiments, the group II metabotropic glutamate receptor agonist, LY354740 was applied to determine the functional presence of these receptors. R-isovaline (1-100mM) decreased the amplitude of ileal muscle contractions and increased tension. RS-baclofen reduced contraction amplitude, but decreased tension. CGP52432 did not prevent the effects of R-isovaline on contraction amplitude, but antagonized effects of RS-baclofen on contraction amplitude. The group II metabotropic glutamate receptor agonist, LY354740, produced no detectable effects on evoked contractions. R-isovaline differed significantly from RS-baclofen in its actions in the guinea pig ileum, indicated in particular by the finding that CGP52432 blocked only the effects of RS-baclofen. The ileal tissue did not respond to a group II metabotropic glutamate receptor agonist, previously shown to co-mediate R-isovaline analgesia. These findings raise the possibility of a novel therapeutic target at unknown receptors for R-isovaline-like compounds in the guinea pig ileum. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of a Potent Free Fatty Acid 1 Receptor Agonist with Low Lipophilicity, Low Polar Surface Area, and Robust in Vivo Efficacy.

PubMed

Hansen, Steffen V F; Christiansen, Elisabeth; Urban, Christian; Hudson, Brian D; Stocker, Claire J; Due-Hansen, Maria E; Wargent, Ed T; Shimpukade, Bharat; Almeida, Reinaldo; Ejsing, Christer S; Cawthorne, Michael A; Kassack, Matthias U; Milligan, Graeme; Ulven, Trond

2016-03-24

The free fatty acid receptor 1 (FFA1 or GPR40) is established as an interesting potential target for treatment of type 2 diabetes. However, to obtain optimal ligands, it may be necessary to limit both lipophilicity and polar surface area, translating to a need for small compounds. We here describe the identification of 24, a potent FFA1 agonist with low lipophilicity and very high ligand efficiency that exhibit robust glucose lowering effect.

Involvement of decreased muscarinic receptor function in prepulse inhibition deficits in mice reared in social isolation

PubMed Central

Koda, K; Ago, Y; Yano, K; Nishimura, M; Kobayashi, H; Fukada, A; Takuma, K; Matsuda, T

2011-01-01

BACKGROUND AND PURPOSE We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation. EXPERIMENTAL APPROACH Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists. KEY RESULTS The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M1 receptor antagonist. Activation of M1 receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M1 receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release. CONCLUSIONS AND IMPLICATIONS Galantamine improves PPI deficits of mice reared in social isolation via activation of M1 receptors. Social isolation reduces the muscarinic, especially M1, receptor function and this is involved in PPI deficits. PMID:20958289

TRPV1 channels in the nucleus of the solitary tract mediate thermal prolongation of the LCR in decerebrate piglets.

PubMed

Xia, Luxi; Bartlett, Donald; Leiter, J C

2011-04-30

Elevating body temperature or just the temperature of the dorsal medulla by approximately 2°C prolongs the laryngeal chemoreflex (LCR) in decerebrate neonatal piglets. We tested the hypothesis that transient receptor potential vanilloid 1 (TRPV1) receptors in the nucleus of the solitary tract (NTS) mediate thermal prolongation of the LCR. We studied the effect of a selective TRPV1 receptor antagonist on thermal prolongation of the LCR, and we tested the effect of a TRPV1 agonist on the duration of the LCR under normothermic conditions. We studied 37 decerebrate neonatal piglets between the ages of post-natal days 4 and 7. The TRPV1 receptor antagonist, 5'-iodoresiniferatoxin (65μM/L in 100nL), blocked thermal prolongation of the LCR when injected bilaterally into the region of the NTS. The TRPV1 agonist, resiniferatoxin (0.65-1.0mM/L in 100nL), prolonged the LCR after bilateral injection into the NTS even when the body temperature of each piglet was normal. The effect of the TRPV1 agonists could be blocked by treatment with the GABA(A) receptor antagonist, bicuculline, whether given intravenously (0.3mg/kg) or focally injected bilaterally into the NTS (10mM in 100nL). We conclude that TRPV1 receptors in the NTS mediate thermal prolongation of the LCR. Copyright © 2011 Elsevier B.V. All rights reserved.

Body temperature and cardiac changes induced by peripherally administered oxytocin, vasopressin and the non-peptide oxytocin receptor agonist WAY 267,464: a biotelemetry study in rats

PubMed Central

Hicks, C; Ramos, L; Reekie, T; Misagh, G H; Narlawar, R; Kassiou, M; McGregor, I S

2014-01-01

Background and Purpose There is current interest in oxytocin (OT) as a possible therapeutic in psychiatric disorders. However, the usefulness of OT may be constrained by peripheral autonomic effects, which may involve an action at both OT and vasopressin V1A receptors. Here, we characterized the cardiovascular and thermoregulatory effects of OT, vasopressin (AVP) and the non-peptide OT receptor agonist WAY 267,464 in rats, and assessed the relative involvement of the OT and V1A receptors in these effects. Experimental Approach Biotelemetry in freely moving male Wistar rats was used to examine body temperature and heart rate after OT (0.01 – 1 mg kg−1; i.p.), AVP (0.001 – 0.1 mg kg−1; i.p.) or WAY 267,464 (10 and 100 mg kg−1; i.p.). The actions of the OT receptor antagonist Compound 25 (C25, 5 and 10 mg kg−1) and V1A receptor antagonist SR49059 (1 and 10 mg kg−1) were studied, as well as possible V1A receptor antagonist effects of WAY 267,464. Key Results OT and AVP dose-dependently reduced body temperature and heart rate. WAY 267,464 had similar, but more modest, effects. SR49059, but not C25, prevented the hypothermia and bradycardia induced by OT and AVP. WAY 267,464 (100 mg·kg−1) prevented the effects of OT, and to some extent AVP. Conclusions and Implications Peripherally administered OT and AVP have profound cardiovascular and thermoregulatory effects that appear to principally involve the V1A receptor rather than the OT receptor. Additionally, WAY 267,464 is not a simple OT receptor agonist, as it has functionally relevant V1A antagonist actions. PMID:24641248

Body temperature and cardiac changes induced by peripherally administered oxytocin, vasopressin and the non-peptide oxytocin receptor agonist WAY 267,464: a biotelemetry study in rats.

PubMed

Hicks, C; Ramos, L; Reekie, T; Misagh, G H; Narlawar, R; Kassiou, M; McGregor, I S

2014-06-01

There is current interest in oxytocin (OT) as a possible therapeutic in psychiatric disorders. However, the usefulness of OT may be constrained by peripheral autonomic effects, which may involve an action at both OT and vasopressin V1A receptors. Here, we characterized the cardiovascular and thermoregulatory effects of OT, vasopressin (AVP) and the non-peptide OT receptor agonist WAY 267,464 in rats, and assessed the relative involvement of the OT and V1A receptors in these effects. Biotelemetry in freely moving male Wistar rats was used to examine body temperature and heart rate after OT (0.01 - 1 mg kg(-1); i.p.), AVP (0.001 - 0.1 mg kg(-1); i.p.) or WAY 267,464 (10 and 100 mg kg(-1); i.p.). The actions of the OT receptor antagonist Compound 25 (C25, 5 and 10 mg kg(-1)) and V1A receptor antagonist SR49059 (1 and 10 mg kg(-1)) were studied, as well as possible V1A receptor antagonist effects of WAY 267,464. OT and AVP dose-dependently reduced body temperature and heart rate. WAY 267,464 had similar, but more modest, effects. SR49059, but not C25, prevented the hypothermia and bradycardia induced by OT and AVP. WAY 267,464 (100 mg·kg(-1)) prevented the effects of OT, and to some extent AVP. Peripherally administered OT and AVP have profound cardiovascular and thermoregulatory effects that appear to principally involve the V1A receptor rather than the OT receptor. Additionally, WAY 267,464 is not a simple OT receptor agonist, as it has functionally relevant V1A antagonist actions. © 2014 The British Pharmacological Society.

Functional Selectivity of Allosteric Interactions within G Protein–Coupled Receptor Oligomers: The Dopamine D1-D3 Receptor Heterotetramer

PubMed Central

Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T.; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.

2014-01-01

The dopamine D1 receptor–D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa–induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R–D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. PMID:25097189

Substance P and dopamine interact to modulate the distribution of delta-opioid receptors on cholinergic interneurons in the striatum.

PubMed

Heath, Emily; Chieng, Billy; Christie, Macdonald J; Balleine, Bernard W

2018-05-01

It has been recently demonstrated that predictive learning induces a persistent accumulation of delta-opioid receptors (DOPrs) at the somatic membrane of cholinergic interneurons (CINs) in the nucleus accumbens shell (Nac-S). This accumulation is required for predictive learning to influence subsequent choice between goal-directed actions. The current experiments investigated the local neurochemical events responsible for this translocation. We found that (1) local administration of substance P into multiple striatal sub-territories induced DOPr translocation and (2) that this effect was mediated by the NK1 receptor, likely through its expression on CINs. Interestingly, whereas intrastriatal infusion of the D1 agonist chloro-APB reduced the DOPr translocation on CINs and infusion of the D2 agonist quinpirole had no effect, co-administration of both agonists again generated DOPr translocation, suggesting the effect of the D1 agonist alone was due to receptor internalisation. In support of this, local administration of cocaine was found to increase DOPr translocation as was chloro-APB when co-administered with the DOPr antagonist naltrindole. These studies provide the first evidence of delta-opioid receptor translocation in striatal cholinergic interneurons outside of the accumbens shell and suggest that, despite differences in local striatal neurochemical microenvironments, a similar molecular mechanism - involving an interaction between dopamine and SP signalling via NK1R - regulates DOPr translocation in multiple striatal regions. To our knowledge, this represents a novel mechanism by which DOPr distribution is regulated that may be particularly relevant to learning-induced DOPr trafficking. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

PubMed Central

Maggi, C. A.; Patacchini, R.; Meini, S.; Quartara, L.; Sisto, A.; Potier, E.; Giuliani, S.; Giachetti, A.

1994-01-01

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7518302

The Therapeutic Potential of Nociceptin/Orphanin FQ Receptor Agonists as Analgesics without Abuse Liability

PubMed Central

2012-01-01

Although mu opioid (MOP) receptor agonists are the most commonly used analgesics for the treatment of moderate to severe pain in the clinic, the side effects of MOP agonists such as abuse liability limit their value as a medication. Research to identify novel analgesics without adverse effects is pivotal to advance the health care of humans. The nociceptin/orphanin FQ peptide (NOP) receptor, the fourth opioid receptor subtype, mediates distinctive actions in nonhuman primates which suggests the possibility that activity at this receptor may result in strong analgesia in the absence of virtually all of the side effects associated with MOP agonists. The present review highlights the recent progress of pharmacological studies of NOP-related ligands in primates. Selective NOP agonists, either peptidic or nonpeptidic, produce full analgesia in various assays in primates, when delivered systemically or intrathecally. Yet small molecule NOP agonists do not serve as reinforcers, indicating a lack of abuse liability. Given that NOP agonists have low abuse liability and that coactivation of NOP and MOP receptors produces synergistic antinociception, it is worth developing bifunctional NOP/MOP ligands. The outcomes of these studies and recent developments provide new perspectives to establish a translational bridge for understanding the biobehavioral functions of NOP receptors in primates and for facilitating the development of NOP-related ligands as a new generation of analgesics without abuse liability in humans. PMID:23421672

Activation of Spinal μ- and δ-Opioid Receptors Potently Inhibits Substance P Release Induced by Peripheral Noxious Stimuli

PubMed Central

Beaudry, Hélène; Dubois, Dave; Gendron, Louis

2013-01-01

Over the past few years, δ-opioid receptors (DOPRs) and μ-opioid receptors (MOPRs) have been shown to interact with each other. We have previously seen that expression of MOPR is essential for morphine and inflammation to potentiate the analgesic properties of selective DOPR agonists. In vivo, it is not clear whether MOPRs and DOPRs are expressed in the same neurons. Indeed, it was recently proposed that these receptors are segregated in different populations of nociceptors, with MOPRs and DOPRs expressed by peptidergic and nonpeptidergic fibers, respectively. In the present study, the role and the effects of DOPR- and MOPR-selective agonists in two different pain models were compared. Using preprotachykinin A knock-out mice, we first confirmed that substance P partly mediates intraplantar formalin- and capsaicin-induced pain behaviors. These mice had a significant reduction in pain behavior compared with wild-type mice. We then measured the effects of intrathecal deltorphin II (DOPR agonist) and DAMGO (MOPR agonist) on pain-like behavior, neuronal activation, and substance P release following formalin and capsaicin injection. We found that both agonists were able to decrease formalin- and capsaicin-induced pain, an effect that was correlated with a reduction in the number of c-fos-positive neurons in the superficial laminae of the lumbar spinal cord. Finally, visualization of NK1 (neurokinin 1) receptor internalization revealed that DOPR and MOPR activation strongly reduced formalin- and capsaicin-induced substance P release via direct action on primary afferent fibers. Together, our results indicate that functional MOPRs and DOPRs are both expressed by peptidergic nociceptors. PMID:21917790

Structural basis for molecular recognition at serotonin receptors.

PubMed

Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

2013-05-03

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Comodulation of dopamine and serotonin on prefrontal cortical rhythms: a theoretical study

PubMed Central

Wang, Da-Hui; Wong-Lin, KongFatt

2013-01-01

The prefrontal cortex (PFC) is implicated to play an important role in cognitive control. Abnormal PFC activities and rhythms have been observed in some neurological and neuropsychiatric disorders, and evidences suggest influences from the neuromodulators dopamine (DA) and serotonin (5-HT). Despite the high level of interest in these brain systems, the combined effects of DA and 5-HT modulation on PFC dynamics remain unknown. In this work, we build a mathematical model that incorporates available experimental findings to systematically study the comodulation of DA and 5-HT on the network behavior, focusing on beta and gamma band oscillations. Single neuronal model shows pyramidal cells with 5-HT1A and 2A receptors can be non-monotonically modulated by 5-HT. Two-population excitatory-inhibitory type network consisting of pyramidal cells with D1 receptors can provide rich repertoires of oscillatory behavior. In particular, 5-HT and DA can modulate the amplitude and frequency of the oscillations, which can emerge or cease, depending on receptor types. Certain receptor combinations are conducive for the robustness of the oscillatory regime, or the existence of multiple discrete oscillatory regimes. In a multi-population heterogeneous model that takes into account possible combination of receptors, we demonstrate that robust network oscillations require high DA concentration. We also show that selective D1 receptor antagonists (agonists) tend to suppress (enhance) network oscillations, increase the frequency from beta toward gamma band, while selective 5-HT1A antagonists (agonists) act in opposite ways. Selective D2 or 5-HT2A receptor antagonists (agonists) can lead to decrease (increase) in oscillation amplitude, but only 5-HT2A antagonists (agonists) can increase (decrease) the frequency. These results are comparable to some pharmacological effects. Our work illustrates the complex mechanisms of DA and 5-HT when operating simultaneously through multiple receptors. PMID:23935568

Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

PubMed

Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

2017-06-01

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn 56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

Bile Acid Receptor Agonist GW4064 Regulates PPARγ Coactivator-1α Expression Through Estrogen Receptor-Related Receptor α

PubMed Central

Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D.

2011-01-01

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology. PMID:21493670

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum.

PubMed

Cheng, Frankie H M; Chan, Sze Wa; Rudd, John A

2008-01-01

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.

Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

PubMed

Castelló-Ruiz, María; Salom, Juan B; Fernández-Musoles, Ricardo; Burguete, María C; López-Morales, Mikahela A; Arduini, Alessandro; Jover-Mengual, Teresa; Hervás, David; Torregrosa, Germán; Alborch, Enrique

2016-10-01

We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated primates, the selective 5-hydroxytryptamine 1a agonist (R)-(+)-8-OHDPAT inhibits levodopa-induced dyskinesia but only with\\ increased motor disability.

PubMed

Iravani, Mahmoud M; Tayarani-Binazir, Kayhan; Chu, Wing B; Jackson, Michael J; Jenner, Peter

2006-12-01

5-Hydroxytryptamine 1a (5-HT(1a)) receptor agonists, such as sarizotan and tandospirone, are reported to reduce levodopa-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques and in Parkinson's disease without worsening motor disability. However, these compounds are not specific for 5-HT(1a) receptors and also possess dopamine antagonist actions. We now report on the effects of (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin [(R)-(+)-8-OHDPAT], a selective 5-HT(1a) agonist lacking dopaminergic activity, on motor disability and dyskinesia (chorea and dystonia) in levodopa-primed MPTP-treated common marmosets. Administration of (R)-(+)-8-OHDPAT (0.2, 0.6, and 2.0 mg/kg s.c), in conjunction with levodopa/carbidopa (12.5 mg/kg each p.o.) to levodopa-primed animals, dose-dependently reduced levodopa-induced chorea but did not affect dystonic movements. However, (R)-(+)-8-OHDPAT treatment also reduced locomotor activity and the reversal of motor disability. Administration of (R)-(+)-8-OHDPAT alone had no effects of motor behaviors. The effects of (R)-(+)-8-OHDPAT on levodopa-induced motor behaviors were antagonized by the 5-HT(1a) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY-100635) (1.0 mg/kg s.c.). Administration of (R)-(+)-8-OHDPAT (0.6 mg/kg s.c.) also reduced chorea produced by the administration of the D(2)/D(3) dopamine receptor agonist pramipexole (0.06 mg/kg p.o.) to levodopa-primed MPTP-treated animals. However, again the increase in locomotor activity and reversal of motor disability produced by pramipexole were also inhibited. These data suggest that selective 5-HT(1a) agonists do not provide an effective means of suppressing levodopa-induced dyskinesia, except with worsening of parkinsonism.

Effects of the GLP-1 Agonist Exendin-4 on Intravenous Ethanol Self-Administration in Mice.

PubMed

Sørensen, Gunnar; Caine, S Barak; Thomsen, Morgane

2016-10-01

Glucagon-like peptide 1 (GLP-1) receptor agonists have been shown to decrease ethanol (EtOH) drinking in rodent assays. The GLP-1 system also powerfully modulates food and fluid intake, gastrointestinal functions, and metabolism. To begin to understand the neurobiological mechanisms by which GLP-1 receptor ligands may be able to control EtOH intake, it is important to ascertain whether they can modulate the direct reinforcing effects of EtOH, without the confound of effects on ingestive behaviors generally. We trained experimentally naïve, free-fed C57BL/6J mice to self-administer EtOH intravenously. Once stable EtOH intake was acquired, we tested the effect of acute pretreatment with the GLP-1 receptor agonist Exendin-4. Effect of Exendin-4 on operant behavior reinforced by a palatable liquid food was similarly evaluated as a control. Intravenous EtOH functioned as a positive reinforcer in over half the mice tested. In mice that acquired self-administration, EtOH intake was high, indeed, reaching toxic doses; 3.2 μg/kg Exendin-4 decreased intravenous EtOH intake by at least 70%, but had no significant effect on food-maintained operant responding. This experiment produced 2 main conclusions. First, although technically challenging and yielding only moderate throughput, the intravenous self-administration procedure in mice is feasible, and sensitive to pharmacological manipulations. Second, GLP-1 receptor agonists can powerfully attenuate voluntary EtOH intake by directly modulating the reinforcing effects of EtOH. These findings support the potential usefulness of GLP-1 receptor ligands in the treatment of alcohol use disorder. Copyright © 2016 by the Research Society on Alcoholism.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

PubMed

Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E

2016-11-11

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Prostaglandin E2 regulates melanocyte dendrite formation through activation of PKCζ

PubMed Central

Scott, Glynis; Fricke, Alex; Fender, Anne; McClelland, Lindy; Jacobs, Stacey

2007-01-01

Prostaglandins are lipid signaling intermediates released by keratinocytes in response to ultraviolet irradiation (UVR) in the skin. The main prostaglandin released following UVR is PGE2, a ligand for 4 related G-protein coupled receptors (EP1, EP2, EP3 and EP4). Our previous work established that PGE2 stimulates melanocyte dendrite formation through activation of the EP1 and EP3 receptors. The purpose of the present report is to define the signaling intermediates involved in EP1 and EP3-dependent dendrite formation in human melanocytes. We recently showed that activation of the atypical PKCζ isoform stimulates melanocyte dendricity in response to treatment with lysophosphatidylcholine. We therefore examined the potential contribution of PKCζ activation on EP1 and EP3-dependent dendrite formation in melanocytes. Stimulation of the EP1 and EP3 receptors by selective agonists activated PKCζ, and inhibition of PKCζ activation abrogated EP1 and EP3-receptor mediated melanocyte dendricity. Because of the importance of Rho-GTP binding proteins in the regulation of melanocyte dendricity, we also examined the effect of EP1 and EP3 receptor activation on Rac and Rho activity. Neither Rac nor Rho was activated upon treatment with EP1,3-receptor agonists. We show that melanocytes express only the EP3A1 isoform, but not the EP3B receptor isoform, previously associated with Rho activation, consistent with a lack of Rho stimulation by EP3 agonists. Our data suggest that PKCζ activation plays a predominant role in regulation of PGE2-dependent melanocyte dendricity. PMID:17850789

New-generation 5-HT4 receptor agonists: potential for treatment of gastrointestinal motility disorders.

PubMed

Manabe, Noriaki; Wong, Banny S; Camilleri, Michael

2010-06-01

Gastrointestinal (GI) dysmotility is an important mechanism in functional GI disorders (FGIDs) including constipation, irritable bowel syndrome, functional dyspepsia, and gastroparesis. 5-hydroxytryptamine(4) (5-HT(4)) receptors are targets for the treatment of GI motility disorders. However, older 5-HT(4) receptor agonists had limited clinical success because they were associated with changes in the function of the cardiac HERG potassium channel. We conducted a PubMed search using the following key words alone or in combination: 5-HT(4), safety, toxicity, pharmacokinetics, pharmacodynamics, clinical trial, cardiac, hERG, arrhythmia, potassium current, elderly, prucalopride, ATI-7505, and velusetrag (TD-5108), to review mechanisms of action, clinical efficacy, safety and tolerability of three new-generation 5-HT(4) receptor agonists. Prucalopride, ATI-7505, and velusetrag (TD-5108) are highly selective, high-affinity 5-HT(4) receptor agonists that are devoid of action on other receptors within their therapeutic range. Their efficacy has been demonstrated in pharmacodynamic studies which demonstrate acceleration of colonic transit and, to a variable degree, in clinical trials that significantly relieve chronic constipation. Currently available evidence shows that the new 5-HT(4) receptor agonists have safe cardiac profiles. New-generation 5-HT(4) receptor agonists and future drugs targeting organ-specific splice variants are promising approaches to treat GI dysmotility, particularly colonic diseases.

Arrestin binds to different phosphorylated regions of the thyrotropin-releasing hormone receptor with distinct functional consequences.

PubMed

Jones, Brian W; Hinkle, Patricia M

2008-07-01

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

PubMed Central

Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Campos-Martínez, Gisselle A.; Meizoso-Huesca, Aldo; García-Sáinz, J. Adolfo

2015-01-01

Results The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes. Conclusion Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes. PMID:26473723

Dopamine D1 and D3 Receptors Modulate Heroin-Induced Cognitive Impairment through Opponent Actions in Mice.

PubMed

Zhu, Yongsheng; Wang, Yunpeng; Lai, Jianghua; Wei, Shuguang; Zhang, Hongbo; Yan, Peng; Li, Yunxiao; Qiao, Xiaomeng; Yin, Fangyuan

2017-03-01

Chronic abuse of heroin leads to long-lasting and complicated cognitive impairment. Dopamine receptors are critically involved in the impulsive drug-driven behavior and the altered attention, processing speed, and mental flexibility that are associated with higher relapse rates. However, the effects of the different dopamine receptors and their possible involvement in heroin-induced cognitive impairment remain unclear. The 5-choice serial reaction time task was used to investigate the profiles of heroin-induced cognitive impairment in mice. The expression levels of dopamine D1- and D2-like receptors in the prefrontal cortex, nucleus accumbens, and caudate-putamen were determined. The effects of dopamine receptors on heroin-induced impulsivity in the 5-choice serial reaction time task were examined by agonist/antagonist treatment on D1 or D3 receptor mutant mice. Systemic heroin administration influences several variables in the 5-choice serial reaction time task, most notably premature responses, a measure of motor impulsivity. These behavioral impairments are associated with increased D1 receptor and decreased D3 receptor mRNA and protein levels in 3 observed brain areas. The heroin-evoked increase in premature responses is mimicked by a D1 agonist and prevented by a D1 antagonist or genetic ablation of the D1 receptor gene. In contrast, a D3 agonist decreases both basal and heroin-evoked premature responses, while genetic ablation of the D3 receptor gene results in increased basal and heroin-evoked premature responses. Heroin-induced impulsive behavior in the 5-choice serial reaction time task is oppositely modulated by D1 and D3 receptor activation. The D1 receptors in the cortical-mesolimbic region play an indispensable role in modulating such behaviors. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Novel 5-aryl-1,3-dihydro-indole-2-thiones. potent, orally active progesterone receptor agonists.

PubMed

Fensome, Andrew; Koko, Marci; Wrobel, Jay; Zhang, Puwen; Zhang, Zhiming; Cohen, Jeffrey; Lundeen, Scott; Rudnick, Kelly; Zhu, Yuan; Winneker, Richard

2003-04-07

During the course of our studies on 3,3-disubstituted-5-aryloxindoles derived progesterone receptor (PR) antagonists we discovered that changing the amide funtionality to a thio-amide resulted in compounds displaying potent PR agonist activity. In this communication, the synthesis, structure activity relationships (SAR) and in vivo activity of various 5-arylthio-oxindoles will be discussed.

Agemone mexicana flavanones; apposite inverse agonists of the β2-adrenergic receptor in asthma treatment.

PubMed

Eniafe, Gabriel O; Metibemu, Damilohun S; Omotuyi, Olaposi I; Ogunleye, Adewale J; Inyang, Olumide K; Adelakun, Niyi S; Adeniran, Yakubu O; Adewumi, Bamidele; Enejoh, Ojochenemi A; Osunmuyiwa, Joseph O; Shodehinde, Sidiqat A; Oyeneyin, Oluwatoba E

2018-01-01

Asthma is an inflammatory disease of the airway that poses a major threat to human health. With increase industrialization in the developed and developing countries, the incidence of asthma is on the rise. The β2-adrenergic receptor is an important target in designing anti-asthmatic drugs. The synthetic agonists of the β2-adrenergic receptor used over the years proved effective, but with indispensable side effects, thereby limiting their therapeutic use on a long-term scale. Inverse agonists of this receptor, although initially contraindicated, had been reported to have long-term beneficial effects. Phytochemicals from Agemone mexicana were screened against the human β2-adrenergic receptor in the agonist, inverse agonist, covalent agonist, and the antagonist conformations. Molecular docking of the phyto-constituents showed that the plant constituents bind better to the inverse agonist bound conformation of the protein, and revealed two flavanones; eriodictyol and hesperitin, with lower free energy (ΔG) values and higher affinities to the inverse agonist bound receptor than the co-crystallized ligand. Eriodictyol and hesperitin bind with the glide score of -10.684 and - 9.958 kcal/mol respectively, while the standard compound ICI-118551, binds with glide score of -9.503 kcal/mol. Further interaction profiling at the protein orthosteric site and ADME/Tox screening confirmed the drug-like properties of these compounds.

Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

PubMed

Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

2017-09-01

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

The Toll-Like Receptor 2/6 Agonist, FSL-1 Lipopeptide, Therapeutically Mitigates Acute Radiation Syndrome.

PubMed

Kurkjian, Cathryn J; Guo, Hao; Montgomery, Nathan D; Cheng, Ning; Yuan, Hong; Merrill, Joseph R; Sempowski, Gregory D; Brickey, W June; Ting, Jenny P-Y

2017-12-11

Risks of radiation exposure from nuclear incidents and cancer radiotherapy are undeniable realities. These dangers urgently compel the development of agents for ameliorating radiation-induced injuries. Biologic pathways mediated by myeloid differentiation primary response gene 88 (MyD88), the common adaptor for toll-like receptor (TLR) and Interleukin-1 receptor signaling, are critical for radioprotection. Treating with agonists prior to radiation enhances survival by activating TLR signaling, whereas radiomitigating TLR-activating therapeutics given after exposure are less defined. We examine the radiomitigation capability of TLR agonists and identify one that is superior for its efficacy and reduced toxic consequences compared to other tested agonists. We demonstrate that the synthetic TLR2/6 ligand Fibroblast-stimulating lipopeptide (FSL-1) substantially prolongs survival in both male and female mice when administered 24 hours after radiation and shows MyD88-dependent function. FSL-1 treatment results in accelerated hematopoiesis in bone marrow, spleen and periphery, and augments systemic levels of hematopoiesis-stimulating factors. The ability of FSL-1 to stimulate hematopoiesis is critical, as hematopoietic dysfunction results from a range of ionizing radiation doses. The efficacy of a single FSL-1 dose for alleviating radiation injury while protecting against adverse effects reveals a viable radiation countermeasures agent.

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor α protein

PubMed Central

Fiori, JL; Sanghvi, M; O'Connell, MP; Krzysik-Walker, SM; Moaddel, R; Bernier, M

2011-01-01

BACKGROUND AND PURPOSE AM251 is an inverse agonist of the cannabinoid 1 receptor (CB1R) that can exert ‘off-target’ effects in vitro and in CB1R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function. EXPERIMENTAL APPROACH The various biological functions of AM251 were measured in CB1R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. KEY RESULTS The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα. CONCLUSIONS AND IMPLICATIONS AM251 up-regulates EGFR expression and signalling via a novel non-CB1R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines. PMID:21449913

Link between D sub 1 and D sub 2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seeman, P.; Niznik, H.B.; Guan, H.C.

1989-12-01

Dopamine receptor types D{sub 1} and D{sub 2} can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D{sub 1}-D{sub 2} interaction in homogenized tissue as revealed by ligand binding. D{sub 2} agonists lowered the binding of ({sup 3}H)raclopride to D{sub 2} receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D{sub 1}-selective antagonist SCH 23390 prevented the agonist-induced decrease in ({sup 3}H)raclopride binding to D{sub 2} sites in the striatum but not in the anterior pituitary, which has no D{sub 1} receptors. Conversely, a dopamine-induced reduction in the binding ofmore » ({sup 3}H)SCH 23390 to D{sub 1} receptors could be prevented by the D{sub 2}-selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D{sub 1}-D{sub 2} interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D{sub 2} receptors in the high-affinity state. Thus, the D{sub 1}-D{sub 2} link may be mediated by guanine nucleotide-binding protein components. The link may underlie D{sub 1}-D{sub 2} interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.« less

Pharmacological, neurochemical, and behavioral profile of JB-788, a new 5-HT1A agonist.

PubMed

Picard, M; Morisset, S; Cloix, J F; Bizot, J C; Guerin, M; Beneteau, V; Guillaumet, G; Hevor, T K

2010-09-01

A novel pyridine derivative, 8-{4-[(6-methoxy-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-3-ylmethyl)-amino]-butyl}-8-aza-spiro[4.5]decane-7,9-dione hydrochloride, termed JB-788, was designed to selectively target 5-HT(1A) receptors. In the present study, the pharmacological profile of JB-788 was characterized in vitro using radioligands binding tests and in vivo using neurochemical and behavioural experiments. JB-788 bound tightly to human 5-HT(1A) receptor expressed in human embryonic kidney 293 (HEK-293) cells with a K(i) value of 0.8 nM. Its binding affinity is in the same range as that observed for the (+/-)8-OH-DPAT, a reference 5HT(1A) agonist compound. Notably, JB-788 only bound weakly to 5-HT(1B) or 5-HT(2A) receptors and moreover the drug displayed only weak or indetectable binding to muscarinic, alpha(2), beta(1) and beta(2) adrenergic receptors, or dopaminergic D(1) receptors. JB-788 was found to display substantial binding affinity for dopaminergic D(2) receptors and, to a lesser extend to alpha(1) adrenoreceptors. JB-788 dose-dependently decreased forskolin-induced cAMP accumulation in HEK cells expressing human 5-HT(1A), thus acting as a potent 5-HT(1A) receptor agonist (E(max.) 75%, EC(50) 3.5 nM). JB-788 did not exhibit any D(2) receptor agonism but progressively inhibited the effects of quinpirole, a D(2) receptor agonist, in the cAMP accumulation test with a K(i) value of 250 nM. JB-788 induced a weak change in cAMP levels in mouse brain but, like some antipsychotics, transiently increased glycogen contents in various brain regions. Behavioral effects were investigated in mice using the elevated plus-maze. JB-788 was found to increase the time duration spent by animals in anxiogenic situations. Locomotor hyperactivity induced by methamphetamine in mouse, a model of antipsychotic activity, was dose-dependently inhibited by JB-788. Altogether, these results suggest that JB-788 displays pharmacological properties, which could be of interest in the area of anxiolytic and antipsychotic drugs. Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

PubMed Central

Bagley, Elena E.

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID:25009497

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

PubMed

Bagley, Elena E

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

GW501516, a PPARδ agonist, ameliorates tubulointerstitial inflammation in proteinuric kidney disease via inhibition of TAK1-NFκB pathway in mice.

PubMed

Yang, Xu; Kume, Shinji; Tanaka, Yuki; Isshiki, Keiji; Araki, Shin-ichi; Chin-Kanasaki, Masami; Sugimoto, Toshiro; Koya, Daisuke; Haneda, Masakazu; Sugaya, Takeshi; Li, Detian; Han, Ping; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi; Uzu, Takashi

2011-01-01

Peroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three different isoforms: PPARα, δ and γ. It has been demonstrated that PPARα and γ agonists have renoprotective effects in proteinuric kidney diseases; however, the role of PPARδ agonists in kidney diseases remains unclear. Thus, we examined the renoprotective effect of GW501516, a PPARδ agonist, in a protein-overload mouse nephropathy model and identified its molecular mechanism. Mice fed with a control diet or GW501516-containing diet were intraperitoneally injected with free fatty acid (FFA)-bound albumin or PBS(-). In the control group, protein overload caused tubular damages, macrophage infiltration and increased mRNA expression of MCP-1 and TNFα. These effects were prevented by GW501516 treatment. In proteinuric kidney diseases, excess exposure of proximal tubular cells to albumin, FFA bound to albumin or cytokines such as TNFα is detrimental. In vitro studies using cultured proximal tubular cells showed that GW501516 attenuated both TNFα- and FFA (palmitate)-induced, but not albumin-induced, MCP-1 expression via direct inhibition of the TGF-β activated kinase 1 (TAK1)-NFκB pathway, a common downstream signaling pathway to TNFα receptor and toll-like receptor-4. In conclusion, we demonstrate that GW501516 has an anti-inflammatory effect in renal tubular cells and may serve as a therapeutic candidate to attenuate tubulointerstitial lesions in proteinuric kidney diseases.

GW501516, a PPARδ Agonist, Ameliorates Tubulointerstitial Inflammation in Proteinuric Kidney Disease via Inhibition of TAK1-NFκB Pathway in Mice

PubMed Central

Yang, Xu; Kume, Shinji; Tanaka, Yuki; Isshiki, Keiji; Araki, Shin-ichi; Chin-Kanasaki, Masami; Sugimoto, Toshiro; Koya, Daisuke; Haneda, Masakazu; Sugaya, Takeshi; Li, Detian; Han, Ping; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi; Uzu, Takashi

2011-01-01

Peroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three different isoforms: PPARα, δ and γ. It has been demonstrated that PPARα and γ agonists have renoprotective effects in proteinuric kidney diseases; however, the role of PPARδ agonists in kidney diseases remains unclear. Thus, we examined the renoprotective effect of GW501516, a PPARδ agonist, in a protein-overload mouse nephropathy model and identified its molecular mechanism. Mice fed with a control diet or GW501516-containing diet were intraperitoneally injected with free fatty acid (FFA)-bound albumin or PBS(−). In the control group, protein overload caused tubular damages, macrophage infiltration and increased mRNA expression of MCP-1 and TNFα. These effects were prevented by GW501516 treatment. In proteinuric kidney diseases, excess exposure of proximal tubular cells to albumin, FFA bound to albumin or cytokines such as TNFα is detrimental. In vitro studies using cultured proximal tubular cells showed that GW501516 attenuated both TNFα- and FFA (palmitate)-induced, but not albumin-induced, MCP-1 expression via direct inhibition of the TGF-β activated kinase 1 (TAK1)-NFκB pathway, a common downstream signaling pathway to TNFα receptor and toll-like receptor-4. In conclusion, we demonstrate that GW501516 has an anti-inflammatory effect in renal tubular cells and may serve as a therapeutic candidate to attenuate tubulointerstitial lesions in proteinuric kidney diseases. PMID:21966476

Evidence for inhibitory nicotinic and facilitatory muscarinic receptors in cholinergic nerve terminals of the rat urinary bladder.

PubMed

Somogyi, G T; de Groat, W C

1992-02-01

Cholinergic prejunctional modulatory receptors on parasympathetic nerves in the rat urinary bladder were studied by measuring 3H-acetylcholine (ACh) release in muscle strips from the bladder body. Electrical field stimulation markedly increased 3H-ACh overflow in strips preloaded with 3H-choline. Oxotremorine (1 microM), an M2 receptor agonist and DMPP (10 microM) a nicotinic (N) receptor agonist decreased the release of ACh (50% and 55% respectively); whereas McN-A 343 (50 microM) an M1 receptor agonist increased the release (33%), indicating the presence of three types of modulatory receptors. The anticholinesterase agent, physostigmine in concentrations of 1, 5 and 25 microM and neostigmine (5 microM) increased ACh release (44-710%). However a low concentration of physostigmine (0.05 microM) decreased release. Pirenzepine, an M1 muscarinic antagonist or atropine blocked the increased ACh release in physostigmine-treated strips, but in normal strips pirenzepine did not change release and atropine increased release. McN-A 343 or prolonged application (15 min) of DMPP increased ACh release (376% and 391% respectively) in physostigmine-treated strips. The response to McN-A 343 was blocked by pirenzepine. d-Tubocurarine (DTC), a nicotinic receptor blocker, enhanced ACh release in the presence of physostigmine but proved to be ineffective in normal preparations. These findings suggest that all three cholinergic receptors (M1 facilitatory, N inhibitory and M2 inhibitory) are activated by endogenous ACh in physostigmine treated preparations whereas only M2-inhibitory receptors are activated in normal preparations. It will be important in future studies to determine whether M1 and M2 mechanisms can also be activated under more physiological conditions in the bladder and whether they are present at other cholinergic synapses.

Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

PubMed

Verbitsky, M; Rothlin, C V; Katz, E; Elgoyhen, A B

2000-10-01

The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

Agonist-dependent modulation of G-protein coupling and transduction of 5-HT1A receptors in rat dorsal raphe nucleus.

PubMed

Valdizán, Elsa Maria; Castro, Elena; Pazos, Angel

2010-08-01

5-HT1A receptors couple to different Go/Gi proteins in order to mediate a wide range of physiological actions. While activation of post-synaptic 5-HT1A receptors is mainly related to inhibition of adenylyl cyclase activity, functionality of autoreceptors located in raphe nuclei has been classically ascribed to modifications of the activity of potassium and calcium channels. In order to evaluate the possible existence of agonist-directed trafficking for 5-HT1A autoreceptors in the rat dorsal raphe nucleus, we studied their activation by two agonists with a different profile of efficacy [(+)8-OH-DPAT and buspirone], addressing simultaneously the identification of the specific Galpha subtypes ([35S]GTPgammaS labelling and immunoprecipitation) involved and the subsequent changes in cAMP formation. A significant increase (32%, p<0.05) in (+)8-OH-DPAT-induced [35S]GTPgammaS labelling of immunoprecipitates was obtained with anti-Galphai3 antibodies but not with anti-Galphao, anti-Galphai1, anti-Galphai2, anti-Galphaz or anti-Galphas antibodies. In contrast, in the presence of buspirone, significant [35S]GTPgammaS labelling of immunoprecipitates was obtained with anti-Galphai3 (50%, p<0.01), anti-Galphao (32%, p<0.01) and anti-Galphai2 (29%, p<0.05) antibodies, without any labelling with anti-Galphai1, anti-Galphaz or anti-Galphas. The selective 5-HT1A antagonist WAY 100635 blocked the labelling induced by both agonists. Furthermore, (+)8-OH-DPAT failed to modify forskolin-stimulated cAMP accumulation, while buspirone induced a dose-dependent, WAY 100635-sensitive, inhibition of this response (Imax 30.8+/-4.9, pIC50 5.95+/-0.46). These results demonstrate the existence of an agonist-dependency pattern of G-protein coupling and transduction for 5-HT1A autoreceptors in native brain tissue. These data also open new perspectives for the understanding of the differential profiles of agonist efficacy in pre- vs. post-synaptic 5-HT1A receptor-associated responses.

Structure-activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH(2) at the mouse melanocortin receptors: part 2 modifications at the Phe position.

PubMed

Holder, Jerry Ryan; Bauzo, Rayna M; Xiang, Zhimin; Haskell-Luevano, Carrie

2002-07-04

The melanocortin pathway is an important participant in skin pigmentation, steroidogenesis, obesity, energy homeostasis and exocrine gland function. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). The melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," and it has been well-documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library, based upon the template Ac-His-DPhe-Arg-Trp-NH(2), consisting of 26 members that have been modified at the DPhe(7) position (alpha-MSH numbering) and pharmacologically characterized for agonist and antagonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the identification of the tetrapeptide Ac-His-(pI)DPhe-Arg-Trp-NH(2) that is a full nanomolar agonist at the mMC1 and mMC5 receptors, a mMC3R partial agonist with potent antagonist activity (pA(2) = 7.25, K(i) = 56 nM) and, but unexpectedly, is a potent agonist at the mMC4R (EC(50) = 25 nM). This ligand possesses novel melanocortin receptor pharmacology, as compared to previously reported peptides, and is potentially useful for in vivo studies to differentiate MC3R vs MC4R physiological roles in animal models, such as primates, where "knockout" animals are not viable options. The DNal(2') substitution for DPhe resulted in a mMC3R partial agonist with antagonist activity (pA(2) = 6.5, K(i) = 295 nM) and a mMC4R (pA(2) = 7.8, K(i) = 17 nM) antagonist possessing 60- and 425-fold decreased potency, respectively, as compared with SHU9119 at these receptors. Examination of this DNal(2')-containing tetrapeptide at the F254S and F259S mutant mMC4Rs resulted in agonist activity of this mMC4R tetrapeptide antagonist, similar to that observed for the SHU9119 peptide, supporting our previously proposed hypothesis that the Phe 254 and 259 transmembrane six receptor residues are important for differentiating melanocortin sequence-based MC4R antagonists vs the agouti-related protein (AGRP) sequence-based antagonists.

6'-Guanidinonaltrindole (6'-GNTI) is a G protein-biased κ-opioid receptor agonist that inhibits arrestin recruitment.

PubMed

Rives, Marie-Laure; Rossillo, Mary; Liu-Chen, Lee-Yuan; Javitch, Jonathan A

2012-08-03

κ-Opioid receptor (KOR) agonists do not activate the reward pathway stimulated by morphine-like μ-opioid receptor (MOR) agonists and thus have been considered to be promising nonaddictive analgesics. However, KOR agonists produce other adverse effects, including dysphoria, diuresis, and constipation. The therapeutic promise of KOR agonists has nonetheless recently been revived by studies showing that their dysphoric effects require arrestin recruitment, whereas their analgesic effects do not. Moreover, KOR agonist-induced antinociceptive tolerance observed in vivo has also been proposed to be correlated to the ability to induce arrestin-dependent phosphorylation, desensitization, and internalization of the receptor. The discovery of functionally selective drugs that are therapeutically effective without the adverse effects triggered by the arrestin pathway is thus an important goal. We have identified such an extreme G protein-biased KOR compound, 6'-guanidinonaltrindole (6'-GNTI), a potent partial agonist at the KOR receptor for the G protein activation pathway that does not recruit arrestin. Indeed, 6'-GNTI functions as an antagonist to block the arrestin recruitment and KOR internalization induced by other nonbiased agonists. As an extremely G protein-biased KOR agonist, 6'-GNTI represents a promising lead compound in the search for nonaddictive opioid analgesic as its signaling profile suggests that it will be without the dysphoria and other adverse effects promoted by arrestin recruitment and its downstream signaling.

Dopamine acting at D1-like, D2-like and α1-adrenergic receptors differentially modulates theta and gamma oscillatory activity in primary motor cortex.

PubMed

Özkan, Mazhar; Johnson, Nicholas W; Sehirli, Umit S; Woodhall, Gavin L; Stanford, Ian M

2017-01-01

The loss of dopamine (DA) in Parkinson's is accompanied by the emergence of exaggerated theta and beta frequency neuronal oscillatory activity in the primary motor cortex (M1) and basal ganglia. DA replacement therapy or deep brain stimulation reduces the power of these oscillations and this is coincident with an improvement in motor performance implying a causal relationship. Here we provide in vitro evidence for the differential modulation of theta and gamma activity in M1 by DA acting at receptors exhibiting conventional and non-conventional DA pharmacology. Recording local field potentials in deep layer V of rat M1, co-application of carbachol (CCh, 5 μM) and kainic acid (KA, 150 nM) elicited simultaneous oscillations at a frequency of 6.49 ± 0.18 Hz (theta, n = 84) and 34.97 ± 0.39 Hz (gamma, n = 84). Bath application of DA resulted in a decrease in gamma power with no change in theta power. However, application of either the D1-like receptor agonist SKF38393 or the D2-like agonist quinpirole increased the power of both theta and gamma suggesting that the DA-mediated inhibition of oscillatory power is by action at other sites other than classical DA receptors. Application of amphetamine, which promotes endogenous amine neurotransmitter release, or the adrenergic α1-selective agonist phenylephrine mimicked the action of DA and reduced gamma power, a result unaffected by prior co-application of D1 and D2 receptor antagonists SCH23390 and sulpiride. Finally, application of the α1-adrenergic receptor antagonist prazosin blocked the action of DA on gamma power suggestive of interaction between α1 and DA receptors. These results show that DA mediates complex actions acting at dopamine D1-like and D2-like receptors, α1 adrenergic receptors and possibly DA/α1 heteromultimeric receptors to differentially modulate theta and gamma activity in M1.

The opioid receptor triple agonist DPI-125 produces analgesia with less respiratory depression and reduced abuse liability.

PubMed

Yi, Shou-Pu; Kong, Qing-Hong; Li, Yu-Lei; Pan, Chen-Ling; Yu, Jie; Cui, Ben-Qiang; Wang, Ying-Fei; Wang, Guan-Lin; Zhou, Pei-Lan; Wang, Li-Li; Gong, Ze-Hui; Su, Rui-Bin; Shen, Yue-Hai; Yu, Gang; Chang, Kwen-Jen

2017-07-01

Opioid analgesics remain the first choice for the treatment of moderate to severe pain, but they are also notorious for their respiratory depression and addictive effects. This study focused on the pharmacology of a novel opioid receptor mixed agonist DPI-125 and attempted to elucidate the relationship between the δ-, μ- and κ-receptor potency ratio and respiratory depression and abuse liability. Five diarylmethylpiperazine compounds (DPI-125, DPI-3290, DPI-130, KUST202 and KUST13T02) were selected for this study. PKA fluorescence redistribution assays in CHO cells individually expressing δ-, μ- or κ-receptors were used to measure the agonist potency. The respiratory safety profiles were estimated in rats by the ratio of ED 50 (pCO 2 increase)/ED 50 (antinociception). The abuse liability of DPI-125 was evaluated with a self-administration model in rhesus monkeys. The observed agonist potencies of DPI-125 for δ-, μ- and κ-opioid receptors were 4.29±0.36, 11.10±3.04, and 16.57±4.14 nmol/L, respectively. The other four compounds were also mixed agonists with varying potencies. DPI-125 exhibited a high respiratory safety profile, clearly related to its high δ-receptor potency. The ratio of the EC 50 potencies for the μ- and δ-receptors was found to be positively correlated with the respiratory safety ratio. DPI-125 has similar potencies for μ- and κ-receptors, which is likely the reason for its reduced abuse potential. Our results demonstrate that the opioid receptor mixed agonist DPI-125 is safer and less addictive than traditional μ-agonist analgesics. These findings suggest that the development of δ>μ∼κ opioid receptor mixed agonists is feasible, and such compounds could represent a promising class of potent analgesics with wider therapeutic windows.

The Endocannabinoid System as a Target for Treatment of Breast Cancer

DTIC Science & Technology

2010-08-01

psychoactive constituent of marijuana (Gaoni and Mechoulam, 1964), as well as other naturally occurring and synthetically derived cannabinoids bind to and...the primary psychoactive constituent present in marijuana , and WIN55,212-2, a highly potent, full CB1 receptor agonist. Female mice implanted with...potent and highly efficacious synthetic cannabinoid receptor agonist originally developed as a nonsteroidal anti-inflammatory drug (Ward et al., 1991

Involvement of μ- and κ-, but not δ-, opioid receptors in the peristaltic motor depression caused by endogenous and exogenous opioids in the guinea-pig intestine

PubMed Central

Shahbazian, Anaid; Heinemann, Akos; Schmidhammer, Helmut; Beubler, Eckhard; Holzer-Petsche, Ulrike; Holzer, Peter

2002-01-01

Opiates inhibit gastrointestinal propulsion, but it is not clear which opioid receptor types are involved in this action. For this reason, the effect of opioid receptor – selective agonists and antagonists on intestinal peristalsis was studied.Peristalsis in isolated segments of the guinea-pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves.μ-Opioid receptor agonists (DAMGO, morphine), κ-opioid receptor agonists (ICI-204,448 and BRL-52,537) and a δ-opioid receptor agonist (SNC-80) inhibited peristalsis in a concentration-related manner as deduced from a rise of the peristaltic pressure threshold (PPT) and a diminution of peristaltic effectiveness.Experiments with the δ-opioid receptor antagonists naltrindole (30 nM) and HS-378 (1 μM), the κ-opioid receptor antagonist nor-binaltorphimine (30 nM) and the μ-opioid receptor antagonist cyprodime (10 μM) revealed that the antiperistaltic effect of ICI-204,448 and BRL-52,537 was mediated by κ-opioid receptors and that of morphine and DAMGO by μ-opioid receptors. In contrast, the peristaltic motor inhibition caused by SNC-80 was unrelated to δ-opioid receptor activation.Cyprodime and nor-binaltorphimine, but not naltrindole and HS-378, were per se able to stimulate intestinal peristalsis as deduced from a decrease in PPT.The results show that the neural circuits controlling peristalsis in the guinea-pig small intestine are inhibited by endogenous and exogenous opioids acting via μ- and κ-, but not δ-, opioid receptors. PMID:11834622

Kindling and withdrawal changes at the benzodiazepine receptor.

PubMed

Little, H J; Nutt, D J; Taylor, S C

1987-01-01

Drugs acting at benzodiazepine receptors can have two types of pharmacological profile: benzodiazepine agonists are anxiolytic, anticonvulsant and sedative, whilst benzo diazepine inverse agonists cause anxiety and convulsions. In 1982 we showed that a benzo diazepine antagonist, Ro 15-1788, prevented the effects of both types of compound at doses without intrinsic activity in the tests used. We put forward the hypothesis that the benzo diazepine receptor complex could undergo two possible conformational changes, resulting in increases (benzodiazepine agonists) or decreases (benzodiazepine inverse agonists) in the effects of the inhibitory transmitter γ-aminobutyric acid (GABA). This concept has been widely accepted. We have now studied the effects of inverse agonists after chronic treatment with inverse agonists themselves and with benzodiazepine agonists, in order to see if tolerance develops (as seen with the agonists) or whether an opposite change occurs.

Lesioning of TRPV1 expressing primary afferent neurons prevents PAR-2 induced motility, but not mechanical hypersensitivity in the rat colon.

PubMed

Suckow, S K; Anderson, E M; Caudle, R M

2012-03-01

Proteinase activated receptor 2 (PAR-2) is expressed by many neurons in the colon, including primary afferent neurons that co-express transient receptor potential vanilloid 1 (TRPV1). Activation of PAR-2 receptors was previously found to enhance colonic motility, increase secretion and produce hypersensitivity to mechanical stimuli. This study examined the functional role of TRPV1/PAR-2 expressing neurons that innervate the colon by lesioning TRPV1 bearing neurons with the highly selective and potent TRPV1 agonist resiniferatoxin. Colonic motility in response to PAR-2 activation was evaluated in vitro using isolated segments of descending colon and in vivo using manometry. Colonic mechanical nociceptive thresholds were measured using colorectal distension. Transient receptor potential vanilloid 1 expressing neurons were selectively lesioned with resiniferatoxin. In vitro, the PAR-2 agonists, trypsin and SLIGRL did not alter contractions of colon segments when applied alone, however, the agents enhanced acetylcholine stimulated contraction. In vivo, PAR-2 agonists administered intraluminally induced contractions of the colon and produced hypersensitivity to colorectal distention. The PAR-2 agonist enhancement of colonic contraction was eliminated when TRPV1 expressing neurons were lesioned with resiniferatoxin, but the PAR-2 agonist induced hypersensitivity remained in the lesioned animals. Our findings indicate that TRPV1/PAR-2 expressing primary afferent neurons mediate an extrinsic motor reflex pathway in the colon. These data, coupled with our previous studies, also indicate that the recently described colospinal afferent neurons are nociceptive, suggesting that these neurons may be useful targets for the pharmacological control of pain in diseases such as irritable bowel syndrome. © 2011 Blackwell Publishing Ltd.

The effect of chronic administration of sarizotan, 5-HT1A agonist/D3/D4 ligand, on haloperidol-induced repetitive jaw movements in rat model of tardive dyskinesia.

PubMed

Rosengarten, Helen; Bartoszyk, Gerd D; Quartermain, David; Lin, Yan

2006-03-01

Dyskinesia is the most troublesome side effect in long-term treatment of both Parkinson's disease (PD) and schizophrenia. The 5-HT1A agonist and D3/D4 ligand sarizotan [Bartoszyk, G.D., van Amsterdam, C., Greiner, H.E., Rautenberg, W., Russ, H., Seyfried, C.A., 2004. Sarizotan, a serotonin 5-HT1A receptor agonist and dopamine receptor ligand. 1. Neurochemical profile. J. Neural Transm. 111, 113-126.] is in clinical development for the treatment of PD-associated dyskinesia. Because 5-HT1A agonists are known to counteract antipsychotic-induced motor side effects, sarizotan was investigated for its effects in two rat models of tardive dyskinesia (TD). The acute administration of sarizotan (0.17-13.5 mg/kg i.p.) reduced episodes of SKF 38393-induced repetitive jaw movements (RJM) in rats with a maximal effect at 1.5 mg/kg. In a chronic study, sarizotan (0.04-9 mg/kg/day), administered in the drinking water for 7 weeks during withdrawal from chronic haloperidol treatment (1.5 mg/kg/day), dose-dependently reversed haloperidol-induced RJM, significant at the doses of 1.5 and 9 mg/kg. Agonism at 5-HT1A receptors may be mediating the inhibitory effect of sarizotan on RJM in rat models of tardive dyskinesia.

An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

PubMed

Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

2014-10-01

In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Active-State Model of a Dopamine D2 Receptor - Gαi Complex Stabilized by Aripiprazole-Type Partial Agonists

PubMed Central

Kling, Ralf C.; Tschammer, Nuska; Lanig, Harald; Clark, Timothy; Gmeiner, Peter

2014-01-01

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy. PMID:24932547

β2-Adrenergic Receptor Agonists Inhibit the Proliferation of 1321N1 Astrocytoma CellsS⃞

PubMed Central

Toll, L.; Jimenez, L.; Waleh, N.; Jozwiak, K.; Woo, A.Y.-H.; Xiao, R.-P.; Bernier, M.

2011-01-01

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses β2-adrenergic receptors (β2-ARs) that are coupled to Gs activation and consequent cAMP production. Experiments were conducted to determine whether the β2-AR agonist (R,R′)-fenoterol and other β2-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize β2-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R′)-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [3H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of β2-AR as determined by receptor binding. (R,R′)-fenoterol and other β2-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [3H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [3H]thymidine incorporation. U118 cells have a reduced number of β2-ARs and a concomitant reduction in the ability of β2-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of β2-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain β2-ARs to a greater extent than whole brain, (R,R′)-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine β2-AR expression. PMID:21071556

Stereochemistry of an agonist determines coupling preference of beta2-adrenoceptor to different G proteins in cardiomyocytes.

PubMed

Woo, Anthony Yiu-Ho; Wang, Tian-Bing; Zeng, Xiaokun; Zhu, Weizhong; Abernethy, Darrell R; Wainer, Irving W; Xiao, Rui-Ping

2009-01-01

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(2)-adrenoceptor, selectively activates G(s) protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G(s) signaling, their S,R isomers were able to activate both G(s) and G(i) proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G(s),G(i2), and G(i3) proteins. The inefficient G(i) signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the beta(2)-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.

Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

PubMed Central

Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

2016-01-01

NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies.

PubMed

Grujic, Ognjen; Stevens, Jennitte; Chou, Robert Y-T; Weiszmann, Jennifer V; Sekirov, Laura; Thomson, Christy; Badh, Anita; Grauer, Stephanie; Chan, Brian; Graham, Kevin; Manchulenko, Kathy; Dillon, Thomas M; Li, Yang; Foltz, Ian N

2017-05-13

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Novel Synthetic Toll-like Receptor 2 Agonists by High Throughput Screening*

PubMed Central

Guan, Yue; Omueti-Ayoade, Katherine; Mutha, Sarita K.; Hergenrother, Paul J.; Tapping, Richard I.

2010-01-01

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit. PMID:20504771

Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

PubMed

Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

2016-01-01

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses. Copyright © 2016 the American Physiological Society.

Troglitazone stimulates {beta}-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1{sub A} receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilley, Douglas G., E-mail: douglas.tilley@jefferson.edu; Center for Translational Medicine, Thomas Jefferson University; Nguyen, Anny D.

2010-06-11

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR{gamma}-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR{gamma} activity, thus we hypothesized that a PPAR{gamma} agonist may exert physiologic effects via the angiotensin II type 1{sub A} receptor (AT1{sub A}R). In AT1{sub A}R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR{gamma} agonist troglitazone (Trog) enhanced AT1{sub A}R internalization and recruitment of endogenous {beta}-arrestin1/2 ({beta}arr1/2) to the AT1{sub A}R. A fluorescence assay to measure diacylglycerolmore » (DAG) accumulation showed that although Ang II induced AT1{sub A}R-G{sub q} protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of {beta}arr1/2 was selective to AT1{sub A}R as the response was prevented by an ARB- and Trog-mediated {beta}arr1/2 recruitment to {beta}1-adrenergic receptor ({beta}1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be {beta}arr2-dependent, as cardiomyocytes isolated from {beta}arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR{gamma} agonist Trog acts at the AT1{sub A}R to simultaneously block G{sub q} protein activation and induce the recruitment of {beta}arr1/2, which leads to an increase in cardiomyocyte contractility.« less

Scalable synthesis and isolation of the four stereoisomers of methyl 1-amino-3-(4-bromophenyl)cyclopentanecarboxylate, useful intermediates for the synthesis of S1P1 receptor agonists.

PubMed

Wallace, Grier A; Gordon, Thomas D; Hayes, Martin E; Konopacki, Donald B; Fix-Stenzel, Shannon R; Zhang, Xiaolei; Grongsaard, Pintipa; Cusack, Kevin P; Schaffter, Lisa M; Henry, Rodger F; Stoffel, Robert H

2009-07-03

The individual isomers of methyl 1-amino-3-(4-bromophenyl)cyclopentanecarboxylate are useful intermediates for the synthesis of S1P1 receptor agonists. Herein we describe a scalable synthesis and isolation of each of the four stereoisomers of this compound in gram quantities with >98% ee and de. The utility of this approach is demonstrated by the synthesis of ((1R,3R)-1-amino-3-(4-octylphenyl)cyclopentyl)methanol in 7 steps, 11% overall yield, and >98% ee and de.

Quantitative Phosphoproteomics Dissection of Seven-transmembrane Receptor Signaling Using Full and Biased Agonists*

PubMed Central

Christensen, Gitte L.; Kelstrup, Christian D.; Lyngsø, Christina; Sarwar, Uzma; Bøgebo, Rikke; Sheikh, Søren P.; Gammeltoft, Steen; Olsen, Jesper V.; Hansen, Jakob L.

2010-01-01

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. “Biased agonists” with intrinsic “functional selectivity” that simultaneously blocks Gαq protein activity and activates G protein-independent pathways of the AT1R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT1R agonist angiotensin II and the biased agonist [Sar1,Ile4,Ile8]angiotensin II (SII angiotensin II), which only activates the Gαq protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT1R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Gαq protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Gαq-dependent and -independent AT1R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gαq protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future. PMID:20363803

Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.

PubMed

Schachter, J B; Wolfe, B B

1992-03-01

The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin.

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

PubMed Central

Garzón, Javier; de la Torre-Madrid, Elena; Rodríguez-Muñoz, María; Vicente-Sánchez, Ana; Sánchez-Blázquez, Pilar

2009-01-01

Background Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated. Results Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids. Conclusion In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs. PMID:19284549

Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

PubMed

Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Alfonzo-Méndez, Marco A; Pupo, André S; García-Sáinz, J Adolfo

2017-12-01

In LNCaP cells that stably express α 1A -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α 1A -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α 1A -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α 1A -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. Copyright © 2017 Elsevier B.V. All rights reserved.

Involvement of the Calcium-sensing Receptor in Human Taste Perception

PubMed Central

Ohsu, Takeaki; Amino, Yusuke; Nagasaki, Hiroaki; Yamanaka, Tomohiko; Takeshita, Sen; Hatanaka, Toshihiro; Maruyama, Yutaka; Miyamura, Naohiro; Eto, Yuzuru

2010-01-01

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca2+, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception. PMID:19892707

Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes.

PubMed

Dalrymple, Matthew B; Jaeger, Werner C; Eidne, Karin A; Pfleger, Kevin D G

2011-05-13

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.