Mouse Brain Membranes Research Articles

Cycloxaprid Insecticide: Nicotinic Acetylcholine Receptor Binding Site and Metabolism

Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

Physical Presence of Nor-Binaltorphimine in Mouse Brain over 21 Days after a Single Administration Corresponds to Its Long-Lasting Antagonistic Effect onκ-Opioid Receptors

In the mouse 55°C warm-water tail-withdrawal assay, a single administration of nor-binaltorphimine (nor-BNI; 10 mg/kg i.p.) antagonized κ-opioid receptor (KOR) agonist-induced antinociception up to 14 days, whereas naloxone (10 mg/kg i.p.)-mediated antagonism lasted less than 1 day. In saturation binding experiments, mouse brain membranes isolated and washed 1 or 7 (but not 14) days after nor-BNI administration demonstrated a significant time-dependent decrease in maximal KOR agonist [(3)H]U69,593 binding. To determine whether brain concentrations of nor-BNI were sufficient to explain the antagonism of KOR-mediated antinociception, mouse blood and perfused brain were harvested at time points ranging from 30 minutes to 21 days after a single administration and analyzed for the presence of nor-BNI using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Nor-BNI was detected in the perfused brain homogenate up to 21 days after administration (30 nmol i.c.v. or 10 mg/kg i.p.). Subsequent experiments in which nor-BNI was administered at doses estimated from the amounts detected in the brain homogenates isolated from pretreated mice over time demonstrated significant antagonism of U50,488 antinociception in a manner consistent with the magnitude of observed KOR antagonism. The dose (1.4 nmol) approximating the lowest amount of nor-BNI detected in brain on day 14 did not antagonize U50,488-induced antinociception, consistent with the absence of U50,488 antagonism observed in vivo at this time point after pretreatment. Overall, the physical presence of nor-BNI in the mouse brain paralleled its in vivo pharmacological profile, suggesting physicochemical and pharmacokinetic properties of nor-BNI may contribute to the prolonged KOR antagonism.

Receptor-type Protein-tyrosine Phosphatase ζ Is a Functional Receptor for Interleukin-34

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

Identification and Characterization of Membrane Proteins from Mouse Brain Tissue

Membrane proteins are attractive targets for proteomics research because of their vital roles in numerous processes of the cell including: cell adhesion, immune response, metabolism and signal transduction... At the start of proteomics research, two-dimensional gel electrophoresis (2D-PAGE) was routinely used to separate complex proteomic samples. However, this method faces difficulties in separating membrane proteins due to their hydrophobicity. In this study, mouse brain membrane fractions were prepared using carbonate extraction and ultracentrifugation. The separation and identification of the membrane proteins by using a gel-based approach in combination with two-dimensional nano liquid chromatography coupled online with tandem mass spectrometry (2DNanoLC-Q-TOF-MS/MS) were presented. In total, 298 identified membrane proteins from mouse brain tissues were verified and predicted by UniProt database, SOSUI and TMHMM algorithms. Among them, 129 (43.3%) proteins that have at least one transmembrane domain were predicted by SOSUI and TMHMM. Furthermore, the function, subcellular location and hydrophobicity value of the identified membrane proteins were also categorized.

Interaction of Voltage-gated Sodium Channel Nav1.6 (SCN8A) with Microtubule-associated Protein Map1b

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.

Identification of Membrane-bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16) as the Non-angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding Site

Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [35S]GTPγS functional assays were performed in the presence of putative δ1-(DPDPE: agonist, BNTX: antagonist), δ2-(agonist: deltorphin II, Ile5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ2- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with KD=0.18nM and receptor densities of 102.7fmol/mg protein in mouse brain membranes. The binding site distribution of the [3H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [3H]Ile5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49±0.06 and 0.30±0.01nM potency against DPDPE and deltorphin II in the [35S]GTPγS functional assay, respectively. The rank order of potency of putative δ1- and δ2-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ1- and δ2-opioid receptors could not be unequivocally distinguished in vitro.

Selective inhibition of [3H]lysergic acid diethylamide binding to mouse brain membranes by ethanol.

Selective inhibition of [3H]lysergic acid diethylamide binding to mouse brain membranes by ethanol.

Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Regulation of CaV1.2 channels in cardiac myocytes by the β-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the β-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Chiral Aryloxyalkylamines: Selective 5‐HT1B/1D Activation and Analgesic Activity

A series of chiral 2,3-dichlorophenoxy and 1-naphthyloxy alkylamines were synthesized, and their binding affinities towards 5-HT(1D) and h5-HT(1B) receptors were evaluated. In the naphthyloxy series, the (R)-prolinol derivative was the most selective 5-HT(1D) ligand, while (S)-N-methyl-2-(1-naphthyloxy)propan-1-amine showed the highest selectivity for h5-HT(1B). Both compounds performed as 5-HT(1D) agonists in the isolated guinea pig assay and showed higher analgesic activity than both sumatriptan and the achiral analogue 20 b in the mouse hot-plate test. Neither ligand displayed any affinity for nicotinic ACh receptors present in mouse brain membranes, thus indicating that their analgesic activity does not arise through interaction with these receptors.

σ1 Receptor Modulation of G-Protein-Coupled Receptor Signaling: Potentiation of Opioid Transduction Independent from Receptor Binding

sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling.

Simultaneous Characterization of Glyco- and Phosphoproteomes of Mouse Brain Membrane Proteome with Electrostatic Repulsion Hydrophilic Interaction Chromatography

Characterization of glyco- and phosphoproteins as well as their modification sites poses many challenges, the greatest being loss of their signals during mass spectrometric detection due to substoichiometric amounts and the ion suppression effect caused by peptides of high abundance. We report here an optimized protocol using electrostatic repulsion hydrophilic interaction chromatography for the simultaneous enrichment of glyco- and phosphopeptides from mouse brain membrane protein digest. With this protocol, we successfully identified 544 unique glycoproteins and 922 glycosylation sites, which were significantly higher than those from the commonly used hydrazide chemistry method (192 glycoproteins and 345 glycosylation sites). Moreover, a total of 383 phosphoproteins and 915 phosphorylation sites were recovered from the sample, suggesting that this protocol has the potential to enrich both glycopeptides and phosphopeptides simultaneously. Of the total 995 glycosylation sites identified from both methods, 96% were considered new as they were either annotated as putative or not documented in the newly released Swiss-Prot database. Thus, this study could be of significant value in complementing the current glycoprotein database and provides a unique opportunity to study the complex interaction of two different post-translational modifications in health and disease without being affected by interexperimental variations.

Evidence that the plant cannabinoid cannabigerol is a highly potent α2‐adrenoceptor agonist and moderately potent 5HT1A receptor antagonist

Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Delta(9)-tetrahydrocannabinol, cannabidiol and Delta(9)-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor. The [(35)S]GTPgammaS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [(3)H]CP55940 from mouse CB(1) and human CB(2) cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated. In the brain membrane experiments, cannabigerol behaved as a potent alpha(2)-adrenoceptor agonist (EC(50)= 0.2 nM) and antagonized the 5-HT(1A) receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K(B)= 51.9 nM). At 10 microM, it also behaved as a CB(1) receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be alpha(2)-adrenoceptor-mediated (EC(50)= 72.8 nM) and displayed significant affinity for mouse CB(1) and human CB(2) receptors. This investigation has provided the first evidence that cannabigerol can activate alpha(2)-adrenoceptors, bind to cannabinoid CB(1) and CB(2) receptors and block CB(1) and 5-HT(1A) receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [(35)S]GTPgammaS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.

KCNE1 and KCNE3 β-Subunits Regulate Membrane Surface Expression of Kv12.2 K+ Channels In Vitro and Form a Tripartite Complex In Vivo

Voltage-gated potassium channels that activate near the neuronal resting membrane potential are important regulators of excitation in the nervous system, but their functional diversity is still not well understood. For instance, Kv12.2 (ELK2, KCNH3) channels are highly expressed in the cerebral cortex and hippocampus, and although they are most likely to contribute to resting potassium conductance, surprisingly little is known about their function or regulation. Here we demonstrate that the auxiliary MinK (KCNE1) and MiRP2 (KCNE3) proteins are important regulators of Kv12.2 channel function. Reduction of endogenous KCNE1 or KCNE3 expression by siRNA silencing, significantly increased macroscopic Kv12.2 currents in Xenopus oocytes by around 4-fold. Interestingly, an almost 9-fold increase in Kv12.2 currents was observed with the dual injection of KCNE1 and KCNE3 siRNA, suggesting an additive effect. Consistent with these findings, over-expression of KCNE1 and/or KCNE3 suppressed Kv12.2 currents. Membrane surface biotinylation assays showed that surface expression of Kv12.2 was significantly increased by KCNE1 and KCNE3 siRNA, whereas total protein expression of Kv12.2 was not affected. KCNE1 and KCNE3 siRNA shifted the voltages for half-maximal activation to more hyperpolarized voltages, indicating that KCNE1 and KCNE3 may also inhibit activation gating of Kv12.2. Native co-immunoprecipitation assays from mouse brain membranes imply that KCNE1 and KCNE3 interact with Kv12.2 simultaneously in vivo, suggesting the existence of novel KCNE1-KCNE3-Kv12.2 channel tripartite complexes. Together these data indicate that KCNE1 and KCNE3 interact directly with Kv12.2 channels to regulate channel membrane trafficking.

Characterization of the brain-specific non-AT 1, non-AT 2 angiotensin binding site in the mouse

In the present study the existence of a non-AT 1, non-AT 2 angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT 1, non-AT 2 angiotensin binding site was similar to the rat brain: Sar 1-Ile 8-Ang II > Ang III ≥ Ang II > Ang I> p-aminophenylalanine 6 Ang II> CGP42112 >> Ang IV > Ang 1–7 ≅ shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K i values > 10 µM, while substance P and VIP had K i values of ~ 2 µM. The non-AT 1, non-AT 2 angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [ 125I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT 1a, AT 1b, AT 2, Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT 1, AT 2, AT 4 and Ang 1–7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study.

A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2‐Arachidonoylglycerol

The endocannabinoid system is the signaling network hijacked by the active component of marijuana, tetrahydrocannibinol (THC), which elicits its effects by activating the cannabinoid (CB) receptors. The endogenous ligands of the CB receptors (“endocannabinoids”) include the lipid transmitters anandamide and 2‐arachidonoyl glycerol (2‐AG). Endocannabinoids have been implicated a wide range of physiological processes including appetite, pain, anxiety and cognition. Endocannabinoid levels in the central nervous system are tightly controlled by enzymatic biosynthesis and degradation. Inactivation of anandamide by fatty acid amide hydrolase (FAAH) has been well characterized, but less is known about the degradation of 2‐AG, which can be hydrolyzed by multiple enzymes in vitro including FAAH and monoacylglyceride lipase (MAGL). We adopted a functional proteomic strategy including activity‐based protein profiling (ABPP) and pharmacological inhibition studies to comprehensively profile 2‐AG hydrolases in the mouse brain. Our results reveal that ~85% of 2‐AG hydrolase activity in mouse brain membranes is performed by MAGL, with the remaining ~15% being catalyzed by two previously uncharacterized enzymes ABHD6 and ABHD12. We determined that MGL, ABHD6, and ABHD12 occupy distinct subcellular localizations, suggesting that they may be responsible for regulating distinct pools of 2‐AG in the nervous system. This work was supported by the National Institutes of Health grants DA015197 and DA017259 (B.F.C), a Fletcher Jones Foundation Scholarship (J.L.B.), the Daniel Koshland Fellowship in Enzyme Biochemistry (G.M.S.), the Skaggs Institute for Chemical Biology, and the Helen L. Dorris Institute for the Study of Neurological and Psychiatric Disorders of Children and Adolescents.

Pharmacological properties of a novel nociceptin/orphanin FQ receptor agonist, 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1 H-benzimidazole, with anxiolytic potential

We have characterized the pharmacological properties of the novel nociceptin/orphanin FQ peptide receptor (NOP receptor) agonist, 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1 H-benzimidazole (PCPB). PCPB bound to the NOP receptor in mouse brain membranes ( Ki = 0.12 nM) and to recombinant human NOP receptor ( Ki = 2.1 nM). PCPB showed full agonism for the NOP receptor in isolated mouse vas deferens with a maximal effect and high potency that were similar to the pharmacological profile observed for nociceptin/orphanin FQ (N/OFQ) (pD 2: 6.9 ± 0.2; 95 ± 2% activity). Orally administered PCPB (30 mg/kg) penetrated well into the brains of the mice. PCPB exhibited an anxiolytic activity in mice subjected to the Vogel conflict test that was comparable to the maximal response induced by diazepam, a representative anxiolytic agent. The anxiolytic effect of PCPB was dose-dependently blocked by the NOP receptor antagonist, J-113397, demonstrating that this effect was mediated by the NOP receptor agonist activity. Behavioral studies in mice also showed that PCPB prolonged the pentobarbital-induced sleeping time but did not cause muscle relaxation at the oral anxiolytic dose of 30 mg/kg. Unlike diazepam, however, these central effects of PCPB were weak. Our results indicate that PCPB is a potent anxiolytic agent with agonistic activities for the NOP receptor.

Memantine prevents MDMA-induced neurotoxicity

MDMA (ecstasy) is an illicit drug causing long-term neurotoxicity. Previous studies demonstrated the interaction of MDMA with alpha-7 nicotinic acetylcholine receptor (nAChR) in mouse brain membranes and the involvement of alpha-7 nicotinic acetylcholine receptors (nAChR) in dopaminergic neurotoxicity induced by MDMA in mice. The aim of the present study was to investigate the utility of memantine (MEM), an alpha-7 nAChR antagonist used for treatment of Alzheimer's disease patients, to prevent neurotoxicity induced by MDMA in rats and the oxidative effect of this amphetamine derivative in mice striatal synaptosomes. In isolated mouse striatal synaptosomes (an in vitro model of MDMA neurotoxicity of dopaminergic origin), MDMA (50 μM)-induced reactive oxygen species (ROS) production that was fully inhibited by MEM (0.3 μM). This effect of MEM was fully prevented by PNU 282987 (0.5 μM), a specific agonist of alpha-7 nAChR. The preventive effect of MEM on this oxidative effect can be attributed to a direct antagonism between MDMA (acting probably as agonist) and MEM (acting as antagonist) at the alpha-7 nAChR. In Dark Agouti rats (an in vivo model of MDMA neurotoxicity of serotonergic origin), a single dose of MDMA (18 mg/kg) induced persistent hyperthermia, which was not affected by MEM pre-treatment. [ 3H]Paroxetine binding (a marker of serotonergic injury) was measured in the hippocampus of animals killed at 24 h and 7 days after treatment. MDMA induced a significant reduction in [ 3H]paroxetine binding sites at both times of sacrifice that was fully prevented by pre-treatment with MEM. Since previous studies demonstrate that increased glutamate activity is not involved in the neurotoxic action of MDMA, it can be concluded that the effectiveness of MEM against MDMA-induced neurotoxicity would be the result of blockade of alpha-7 nAChR, although an indirect mechanism based on the interplay among the various neurotransmission systems leading to an increase in basal acetylcholine release should also be taken into account.

The psychoactive plant cannabinoid, Δ9‐tetrahydrocannabinol, is antagonized by Δ8‐ and Δ9‐tetrahydrocannabivarin in mice in vivo

Background and purpose:To follow up in vitro evidence that Δ9‐tetrahydrocannabivarin extracted from cannabis (eΔ9‐THCV) is a CB1 receptor antagonist by establishing whether synthetic Δ9‐tetrahydrocannabivarin (O‐4394) and Δ8‐tetrahydrocannabivarin (O‐4395) behave as CB1 antagonists in vivo.Experimental approach:O‐4394 and O‐4395 were compared with eΔ9‐THCV as displacers of [3H]‐CP55940 from specific CB1 binding sites on mouse brain membranes and as antagonists of CP55940 in [35S]GTPγS binding assays performed with mouse brain membranes and of R‐(+)‐WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg−1 (i.v.) Δ9‐tetrahydrocannabinol in mice was then investigated.Key results:O‐4394 and O‐4395 exhibited similar potencies to eΔ9‐THCV as displacers of [3H]‐CP55940 (K i=46.6 and 64.4 nM, respectively) and as antagonists of CP55940 in the [35S]GTPγS binding assay (apparent K B=82.1 and 125.9 nM, respectively) and R‐(+)‐WIN55212 in the vas deferens (apparent K B=4.8 and 3.9 nM respectively). At i.v. doses of 0.1, 0.3, 1.0 and/or 3 mg kg−1 O‐4394 and O‐4395 attenuated Δ9‐tetrahydrocannabinol‐induced anti‐nociception (tail‐flick test) and hypothermia (rectal temperature). O‐4395 but not O‐4394 also antagonized Δ9‐tetrahydrocannabinol‐induced ring immobility. By themselves, O‐4395 and O‐4394 induced ring immobility at 3 or 10 mg kg−1 (i.v.) and antinociception at doses above 10 mg kg−1 (i.v.). O‐4395 also induced hypothermia at 3 mg kg−1 (i.v.) and above.Conclusions and implications:O‐4394 and O‐4395 exhibit similar in vitro potencies to eΔ9‐THCV as CB1 receptor ligands and as antagonists of cannabinoid receptor agonists and can antagonize Δ9‐tetrahydrocannabinol in vivo.British Journal of Pharmacology (2007) 150, 586–594. doi:10.1038/sj.bjp.0707124