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To demonstrate the antinociceptive and antihypersensitivity mechanisms of Cris-104 (1-{2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl}piperidine), a novel selective α β * nicotinic acetylcholine receptor (nAChR) agonist, in rodent acute/inflammatory and chronic pain models. Hot-plate and formalin tests in mice were used to examine Cris-104-induced antinociceptive effects on thermal/inflammatory pain. Cris-104 effects on hypersensitivity, norepinephrine (NE) release in the spinal dorsal horn, and neuronal activity in the locus coeruleus (LC) were examined in rats with lumbar spinal nerve ligation using behavioral, microdialysis, and extracellular recording methods. Cris-104 effects on spontaneous locomotion were examined in an open-field test. Cris-104 induced dose-dependent antinociception effects in hot-plate and formalin tests, and these effects were blocked by the general nAChR antagonist mecamylamine, the selective α β * nAChR antagonist dihydro-beta-erythroidine, and the α -adrenoceptor antagonist yohimbine, but not by the α -adrenoceptor antagonist prazosin. Systemic and spinally perfused Cris-104 increased NE concentrations in microdialysates from the spinal cord in both normal and SNL rats. Systemic Cris-104 increased neuronal activity in the LC of normal rats. Mecamylamine blocked the effects of Cris-104 on spinal NE release and LC neuronal activity. Systemic Cris-104 did not affect locomotor activity significantly. The α β neuronal nAChR agonist, Cris-104, was effective for treatment of pain via descending noradrenergic inhibition of pain signaling.

Rationale Individuals vary in sensitivity to the behavioral effects of nicotine, resulting in differences in vulnerability to nicotine addiction. The role of rearing environment in determining individual sensitivity to nicotine is unclear. The neuropharmacological mechanisms mediating the effect of rearing environment on the behavioral actions of nicotine are also poorly understood. Objectives The contribution of rearing environment in determining the sensitivity to the interoceptive effects of nicotine was determined in rats reared in isolated conditions (IC) or enriched conditions (EC). The role of dopamine receptors and α4β2*-nicotinic acetylcholine (nACh) receptors in mediating the differential effect of IC and EC on the interoceptive action of nicotine was determined. Methods The interoceptive action of nicotine was measured as the discriminative stimulus effect of nicotine. Mecamylamine- and eticlopride-inhibition of the nicotine stimulus were used to examine nACh and dopamine receptors, respectively. α4β2*-nACh receptor expression in the mesolimbic dopamine pathway was determined by quantitative autoradiography of [ 125 I]-epibatidine binding. Results EC-reared rats are less sensitive than IC-reared rats to the discriminative stimulus effects of nicotine at all but maximally effective doses. Mecamylamine inhibited the nicotine stimulus threefold more potently in EC-reared rats (IC 50  = 0.25 mg/kg) compared to IC-reared rats (IC 50  = 0.75 mg/kg); eticlopride inhibition was not different. [ 125 I]-epibatidine binding in the ventral tegmental area of EC-reared rats was reduced (2.8 ± 0.3 fmol) compared to that of IC-reared rats (4.0 ± 0.4 fmol); there was no difference in the nucleus accumbens. Conclusions Rearing environment regulates the sensitivity to the interoceptive effects of nicotine and α4β2*-nACh receptor expression in the mesolimbic dopamine pathway.

Dendrobatid poison frogs sequester alkaloids from an arthropod diet and use them in chemical defense. Alkaloid defenses vary considerably within and among species, with important consequences for the protection they can and do provide against microorganisms and predators. Most of this variation is attributed to differences in frog diet and prey availability, but emerging evidence also suggests that frogs differ in their physiological ability to sequester alkaloids. Epibatidines are one of the most geographically and phylogenetically restricted alkaloid classes in poison frogs, having been found naturally only in two genera of dendrobatids (Epipedobates and Ameerega) from Ecuador and northern Peru. To test the hypothesis that the ability to sequester epibatidine is confined to the lineages Epipedobates and Ameerega, we experimentally administered epibatidine to individuals of five species, representing three different lineages of dendrobatid poison frogs, including those known to possess (Epipedobates anthonyi) and lack (Ranitomeya variabilis, Ranitomeya imitator, Phyllobates vittatus, Dendrobates tinctorius) epibatidines in nature. All five species sequestered epibatidine; however, the percentage sequestered varied significantly across species with Epipedobates and Ranitomeya accumulating about 2.4× more than Phyllobates or Dendrobates. Our results suggest that the absence of epibatidine in certain dendrobatids is not due to the inability of these frogs to sequester epibatidine, but may instead result from differences in prey availability and/or dietary preference. Our finding of differences in the percentage of epibatidine sequestered among species points to the importance that physiological differences in sequestration play in explaining some of the alkaloid variation (including epibatidine) observed among dendrobatid poison frogs.

Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms toward an adaptive peak of chemical defense.

RationaleProlinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4β2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4β2 receptors, but their in vivo effects are unknown.Objectives and methodsAs α4β2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated.ResultsIn comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors.ConclusionsThese behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.

Epibatidine is a natural alkaloid that acts at nicotinic acetylcholine receptors (nAChRs). The present review aims to carefully discuss the affinity of epibatidine and its synthetic derivatives, analogues to nAChRs for α4β2 subtype, pharmacokinetic parameters, and its role in health. Published literature shows a low affinity and lack of binding of epibatidine and its synthetic analogues to plasma proteins, indicating their availability for metabolism. Because of its high toxicity, the therapeutic use of epibatidine is hampered. However, new synthetic analogs endowed from this molecule have been developed, with a better therapeutic window and improved selectivity. All these aspects are also discussed here. On the other hand, many reports are devoted to structure–activity relationships to obtain optically active epibatidine and its analogues, and to access its pharmacological effects. Although pharmacological results are obtained from experimental studies and only a few clinical trials, new perspectives are open for the discovery of new drug therapies.

Background Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor β2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4β2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration–response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the β2 S108C into the α4β2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. Results The introduction of S108C into the α4β2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4β2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. Conclusions The most parsimonious explanation of our current and previous work is that the S108C substitution renders the β2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while β2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4β2 receptor function.

αD-conotoxins are 11 kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein ( Ls -AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls -AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Neuronal nicotinic acetylcholine receptors (nAChRs) of the cholinergic system have been linked to antinociception, and therefore could be an alternative target for pain alleviation. nAChR activity has been shown to be regulated by the nicotinic modulator, lynx1, which forms stable complexes with nAChRs and has a negative allosteric action on their function. The objective in this study was to investigate the contribution of lynx1 to nicotine-mediated antinociception. Lynx1 contribution was investigated by mRNA expression analysis and electrophysiological responses to nicotine in the dorsal raphe nucleus (DRN), a part of the pain signaling pathway. In vivo antinociception was investigated in a test of nociception, the hot-plate analgesia assay with behavioral pharmacology. Lynx1/α4β2 nAChR interactions were investigated using molecular dynamics computational modeling. Nicotine evoked responses in serotonergic and GABAergic neurons in the DRN are augmented in slices lacking lynx1 (lynx1KO). The antinociceptive effect of nicotine and epibatidine is enhanced in lynx1KO mice and blocked by mecamylamine and DHβE. Computer simulations predict preferential binding affinity of lynx1 to the α:α interface that exists in the stoichiometry of the low sensitivity (α4)3(β2)2 nAChRs. Taken together, these data point to a role of lynx1 in mediating pain signaling in the DRN through preferential affinity to the low sensitivity α4β2 nAChRs. This study suggests that lynx1 is a possible alternative avenue for nociceptive modulation outside of opioid-based strategies.