Ventral hippocampal α4β2 nicotinic receptors and chronic nicotine effects on memory

Chronic nicotine administration has been repeatedly shown to facilitate working memory function in rats on the radial-arm maze. The critical neural mechanisms for this effect are still being discovered. The nicotinic nature of the chronic nicotine induced memory improvement is supported by the finding that it is blocked by chronic mecamylamine co-infusion. The hippocampus also appears to be critically important. Hippocampal ibotenic acid lesions block the effect. Within the hippocampus, we have found that the α4β2 nicotinic receptor subtype is involved in memory functioning. Acute ventral hippocampal infusions of the α4β2 nicotinic antagonist dihydro-β-erythroidine (DHβE) significantly decreased working memory performance in the radial-arm maze. The aim of the current study was to determine the importance of α4β2 receptors within the ventral hippocampus for the memory enhancing effects of chronic nicotine treatment. Adult female Sprague–Dawley rats were trained on the 8-arm radial maze and were cannulated bilaterally in the ventral hippocampus. Osmotic minipumps administering chronic nicotine at a rate of 5 mg per kg per day were also implanted in the nicotine treatment rats. Control rats received saline-only minipumps. For a period of 4 weeks after surgery, each rat received bilateral hippocampal infusions of 0, 2, 6 and 18 μg per side of DHβE and tested for memory performance on the radial-arm maze. Radial-arm maze choice accuracy was impaired by acute hippocampal DHβE infusion in a dose-related fashion. This acute hippocampal DHβE-induced choice accuracy impairment was eliminated by chronic systemic nicotine infusion. Chronic nicotine in combination with acute vehicle hippocampal infusion was not seen to alter choice accuracy. Response latency was not found to be altered by acute hippocampal DHβE in the absence of chronic nicotine administration, but it did attenuate the response latency reduction induced by chronic nicotine infusion. Wet dog shakes were not found to be affected by hippocampal DHβE when given without chronic nicotine. Wet dog shakes were significantly increased by chronic nicotine infusion. Intra-hippocampal DHβE significantly potentiated this effect. The results from the current study reinforce the hypothesis that ventral hippocampal α4β2 nicotinic receptors are important for memory function. These receptors may also have a role to play in the development of other aspects of behavior associated with chronic nicotine treatment.     

Antagonism of the anxiolytic effect of nicotine in the dorsal raphé nucleus by dihydro-β-erythroidine

Nicotine has been reported to reduce anxiety in humans and in a number of animal tests. In the social interaction test of anxiety, administration of low doses of nicotine into the dorsal raphé nucleus (DRN) increases the time spent in social interaction without producing accompanying changes in locomotor activity, suggesting that nicotine acts specifically to reduce anxiety in this brain region. The present study examined the ability of the high-affinity competitive nicotinic receptor antagonist dihydro-β-erythroidine hydrobromide (DHβE) to antagonise the anxiolytic effect of nicotine following intra-DRN infusion using the social interaction test. The increase in social interaction observed after administration of nicotine (5 ng) into the DRN was completely reversed by coadministration of 100 ng DHβE. DHβE (100 ng), when administered alone into the DRN, did not modify the time spent in social interaction. However, it did significantly increase locomotor activity, and this effect was not antagonised by coadministration of nicotine (5 ng) into the DRN. Because of the pharmacological profile of DHβE, our results suggest that the anxiolytic effect of nicotine in the DRN is mediated by the 4β2 nicotinic receptor subtype.     

The opioid antagonist naltrexone inhibits activity and alters expression of α7 and α4β2 nicotinic receptors in hippocampal neurons: implications for smoking cessation programs

This study was designed to investigate whether naltrexone, an opioid antagonist that has been evaluated clinically as a co-adjuvant in smoking cessation programs, affects function and expression of neuronal nicotinic receptors (nAChRs). Whole-cell current recordings from rat hippocampal neurons in culture and in slices demonstrated that α7 nAChRs can be inhibited non-competitively by naltrexone (IC₅₀25 μM). The voltage dependence of the effect suggested that naltrexone acts as an open-channel blocker of α7 nAChRs. Naltrexone also inhibited activation of α4β2 nAChRs in hippocampal neurons; however its IC₅₀ was higher (141 μM). At a concentration as high as 300 μM (which is sufficient to block by 100% and 70% the activity of α7 and α4β2 nAChRs, respectively), naltrexone had no effect on kainate and AMPA receptors, blocked by no more than 20% the activity of NMDA and glycine receptors, and reduced by 35% the activity of GABA_A receptors. A 3-day exposure of cultured hippocampal neurons to naltrexone (30 μM) or nicotine (10 μM, a concentration that fully desensitized α7 nAChRs) resulted in a 2-fold increase in the average amplitude of α7 nAChR-subserved currents. Naltrexone did not augment the maximal up-regulation of α7 nAChRs induced by nicotine, indicating that both drugs act via a common mechanism. In addition to increasing α7 nAChRs-mediated responses per neuron, nicotine increased the number of neurons expressing functional non-α7 nAChRs (probably α4β2 nAChRs); this effect was blocked by naltrexone (0.3 and 30 μM). Therefore, naltrexone may affect dependence on cigarette smoking by differentially altering function and expression of α7 and α4β2 nAChRs in the central nervous system.     

Reinforcement enhancing effect of nicotine and its attenuation by nicotinic antagonists in rats

Rationale Recent studies have demonstrated that nicotine can enhance operant responding for other nonpharmacological reinforcing stimuli. However, the nature of the reinforcement-enhancing effect of nicotine remains largely unknown. Objective The present study determined the dose dependency of the ability of nicotine to increase lever-pressing responses maintained by a compound visual stimulus (VS) in rats and examined its sensitivity to pharmacological antagonism of nicotinic acetylcholine receptors (nAChRs). Materials and methods Male Sprague–Dawley rats were trained in daily 1-h sessions to lever press for delivery of a VS (1 s lever light on and 60 s house light off) on a fixed ratio 5 schedule. During these sessions, eight scheduled response-independent intravenous infusions of nicotine (total amount: 0, 0.06, 0.12, 0.24, 0.48 mg kg⁻¹ h⁻¹) were delivered. In pharmacological tests, a nonselective nAChR antagonist mecamylamine, α4β2-selective antagonist dihydro-β-erythroidine (DHβE), and α7-selective antagonist methyllycaconitine (MLA) were administered in different groups of rats 30 min before the session. Results The VS maintained a moderate level of lever-pressing responses and nicotine dose-dependently increased responses for the VS presentations. Preteatment of mecamylamine and DHβE but not MLA significantly attenuated the nicotine-enhanced responding. However, mecamylamine had no effect on responding for the VS in rats that received scheduled saline infusions. Conclusions These results demonstrate dose dependency of the reinforcement-enhancing effect of nicotine and suggest that activation of the α4β2- but not α7-containing nAChRs may mediate this effect.     

Diminished conditioned responding to the nicotine stimulus by antidepressant drugs with differing specificity for the serotonin and norepinephrine transporter

People diagnosed with depression also tend to have a co-morbid nicotine addiction. Thus, there is interest in whether medications used to treat depression alter the effects of nicotine. This study assessed whether the antidepressant drugs citalopram, imipramine, and reboxetine, with differing specificity for the serotonin and norepinephrine transporter, altered responding controlled by the conditional stimulus (CS) effects of nicotine. Rats received intermixed 20-min nicotine (0.4 mg base/kg, SC) and saline sessions. On nicotine sessions, rats had intermittent access to sucrose; no sucrose was available on saline sessions. After discrimination performance stabilized and a nicotine generalization curve (0.025-0.4 mg/kg) was established, the antidepressant drugs were assessed. In these tests, rats were pretreated with citalopram (1-17 mg/kg), imipramine (1-17 mg/kg), or reboxetine (1-30 mg/kg) before the training dose of nicotine and placement in a chamber for a 4-min extinction test. At the higher doses, all three antidepressant drugs blocked responding evoked by the nicotine CS and decreased nicotine-induced hyperactivity. When these higher doses of citalopram, imipramine, and reboxetine were tested alone (no nicotine), they decreased chamber activity and/or dipper entries. Nevertheless, all three drugs produced partial or complete blockade of the CS effects of nicotine at doses that produced no effect on dipper entries or chamber entries. This finding suggests that both neurotransmitters play a role in the CS effects of nicotine and that modifications in these systems by antidepressants may be clinically relevant.     

Evaluation of the role of nicotinic acetylcholine receptor subtypes and cannabinoid system in the discriminative stimulus effects of nicotine in rats

Male Wistar rats were trained to discriminate (−)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025–0.4 mg/kg; combination studies): the ₄β₂ nicotinic acetylcholine receptor subtype antagonist dihydro-β-erythroidine (DHβE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the ₇ nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the ₄β₂ nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB₁ receptor antagonist/partial agonist rimonabant, the cannabinoid CB₂ receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB_1/2 receptor agonists (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive ₇ nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404 + anandamide or URB 597 + anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHβE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404 + anandamide or URB 597 + anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of ₄β₂ (but not of ₇) subunits. Furthermore, we excluded the involvement of either cannabinoid CB₁ and CB₂ receptors or increases in the endocannabinoid tone in the nicotine discrimination.     

Investigation of endocannabinoid modulation of conditioned responding evoked by a nicotine CS and the Pavlovian stimulus effects of CP 55,940 in adult male rats

Rationale  The cannabinoid CB₁ receptor antagonist/inverse agonist rimonabant (SR 141716) has been shown to block reinforcing and rewarding effects of nicotine. Research has not investigated whether the cannabinoid system is involved in the interoceptive stimulus effects of nicotine functioning as a conditional stimulus (CS). Objective  We examined the effects of rimonabant and the CB_1/2 receptor agonist, CP 55,940, on responding evoked by a nicotine CS in rats. Additionally, we determined whether CP 55,940 functioned as a CS or a Pavlovian positive drug feature Materials and methods  Pavlovian discrimination training involved intermixed nicotine (0.2 mg base/kg) and saline sessions with intermittent access to water only on nicotine. Antagonism tests with rimonabant (0.1-3 mg/kg) and substitution tests with CP 55,940 (0.003–0.1 mg/kg) followed. An effective dose of CP 55,940 was tested against the nicotine generalization curve. A separate group received CS training with CP 55,940 (0.01 mg/kg). Two other groups were trained using CP 55,940 (0.01 or 0.03 mg/kg) as a positive drug feature in which a brief light CS signaled access to water only on CP 55,940 sessions Results  Rimonabant blocked nicotine-evoked responding. CP 55,940 partially substituted for nicotine and enhanced responding to lower nicotine doses. Overall, CP 55,940 did not acquire control of conditioned responding in either Pavlovian drug discrimination task Conclusions  The cannabinoid system was involved in the CS effects of nicotine. This finding is counter to the operant drug discrimination research with nicotine as a discriminative stimulus, warranting further research into this possible dissociation.     

Antagonism of the discriminative and aversive stimulus properties of nicotine in C57BL/6J mice

Mice of the C56BL/6J strain were trained to discriminate between nicotine (1.2 mg/kg) and saline in a two-lever drug discrimination procedure under a tandem variable-interval 60 s fixed-ratio 10 schedule of food reinforcement. Mice of the same strain were trained in conditioned taste aversion (CTA) experiments where drinking a saccharin or saline solution was paired with injection of nicotine or vehicle. During testing with both flavours presented simultaneously, a reduction in the intake of the nicotine-paired solution indicated CTA. The nicotine discrimination was acquired successfully and nicotine yielded a steep dose–response curve. The competitive nicotinic antagonist dihydro-β-erythroidine (DHβE, 0.6–3.0 mg/kg) shifted the dose–response for the discriminative stimulus effect of nicotine to the right; the 7 nicotinic receptor antagonist methyllycaconitine (MLA, 1.0–10 mg/kg) had no effect. The mice showed strong CTA to 2.0 mg/kg of nicotine and marginally to 0.6 and 1.2 mg/kg of nicotine. DHβE (3.0–5.6 mg/kg) attenuated the CTA while MLA (1.0–10 mg/kg) had no effect. These studies show that nicotine has discriminative and aversive stimulus properties in C57BL/6J mice and that the effects are mediated primarily by receptors sensitive to DHβE; there was no evidence for the involvement of 7 nicotinic receptors.     

Extinction with varenicline and nornicotine, but not ABT-418, weakens conditioned responding evoked by the interoceptive stimulus effects of nicotine

The interoceptive stimulus effects of nicotine acquire control over behavior. This observation, among others, suggests that the stimulus effects of nicotine are important in the development and tenacity of tobacco dependence. Despite this importance, there has been little research examining whether non-reinforced presentations (extinction) of a ligand that share stimulus effects of nicotine will weaken responding controlled by nicotine. Rats were trained to discriminate nicotine (0.4 mg/kg) from saline using a discriminated goal-tracking task in which nicotine signaled intermittent access to sucrose; sucrose was withheld on saline sessions. Experiment 1 examined substitution for nicotine by ABT-418, nornicotine, epibatidine, varenicline, or cytisine in 4-min extinction tests. Experiments 2–5 [low-dose nicotine (0.05 mg/kg), ABT-418, nornicotine, or varenicline, respectively] examined whether substitution for nicotine would persist if extinction tests were increased to 20 min and repeated daily for 6 days. Finally, generalization of this extinction back to the nicotine training stimulus was assessed. Full substitution in brief 4-min extinction tests was seen for ABT-418, nornicotine, epibatidine, varenicline, and cytisine. Low-dose nicotine, ABT-418, nornicotine, and varenicline, evoked only a partial ‘nicotine-like’ response in the first 20-min extinction test. With repeated extinction, only low-dose nicotine, nornicotine, and varenicline continued to substitute. Extinction with nornicotine and varenicline transferred back to nicotine as indicated by a partial conditioned response to the training stimulus. Interpretations regarding ‘nicotine-like’ effects of a ligand depend on the nature of the test. Understanding the processes mediating transfer of extinction learning with potential pharmacotherapies may reveal new treatment targets.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Selective α7 nicotinic receptor activation by AZD0328 enhances cortical dopamine release and improves learning and attentional processes

AZD0328, a novel spirofuropyridine neuronal nicotinic receptor partial agonist, was used to investigate the role of α7 neuronal nicotinic receptor (NNR) activation in the modulation of midbrain dopamine neuron function, cortical dopamine release and on two behavioral tasks known to be dependent on optimal levels of cortical dopamine. In vivo recordings from area 10 (ventral tegmental area) in rat brain showed an increased firing of putative dopamine neurons in response to low (0.00138 mg/kg) doses of AZD0328. Bursting patterns of dopamine neuron activity remained largely unchanged by application of AZD0328. In vivo microdialysis in awake rats showed an increase in extracellular prefrontal cortical dopamine in response to low doses of AZD0328. Compound-stimulated dopamine release showed an inverted dose effect relation that was maximal at the lowest dose tested (0.00178 mg/kg). Peak extracellular dopamine levels were reached 2 h after dosing with AZD0328. Acquisition of operant responding with delayed reinforcement in rats was dose dependently enhanced by AZD0328 with a plateau effect measured at 0.003 mg/kg. This effect was blocked by pre-treatment of animals with the selective α7 antagonist methyllycaconitine. AZD0328 improved novel object recognition in mice over a broad range of doses (0.00178–1.78 mg/kg) and the compound effect was found to be absent in homozygous α7 KO animals. Together, these data indicate that selective interaction with α7 NNRs by AZD0328 selectively enhances midbrain dopaminergic neuronal activity causing an enhancement of cortical dopamine levels; these neurochemical changes likely, underlie the positive behavioral responses observed in two different animal models. Our results suggest selective α7 NNR agonists may have significant therapeutic utility in neurologic and psychiatric indications where cognitive deficits and dopamine neuron dysfunction co-exist.     

The unusual nature of epibatidine responses at the α4β2 nicotinic acetylcholine receptor

The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat α4β2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the α4β2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat α4β2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.     

Relaxant β-Receptors in the Trachea,but not Prejunctional α2-Receptors in the Tracheal Cholinergic Neurones,are Subjected to Homologous Desensitization

Abstract: Guinea-pigs were pretreated with either isoprenaline, terbutaline or the α₂ agonist B-HT 920 in order to asses the hypothesis that β- and α₂ receptors in trachea are subjected to homologous desensitization. In these experiments the β receptor activity was investigated on tracheal ring preparations contracted with carbacholine. Both in the isoprenaline and the terbutaline pretreated group the relaxant responses to β agonists were diminished. Pretreatment with B-HT 920 did not affect the sensitivity of the trachea to β stimulation. In order to asses the responsiveness of α₂ receptors the trachea was contracted by electrical field stimulation in the presence of propranolol. During these conditions contractions were mediated by activation of cholinergic neurones and inhibitory effects of alpha stimulation were due to inhibition of the cholinergic neurotransmission by stimulation of prejunctional α₂ receptors. In these tests neither isoprenaline, terbutaline nor B-HT 920 pretreatment affected the responsiveness of α stimulation to inhibit the electrically induced contractions.     

Association of β-agonists with corresponding β2- and β1-adrenergic pentapeptide sequences

Synthesized β₁- and β₂-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane α-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the β-adrenoreceptors.     

Selectivity of ABT-089 for α4β2* and α6β2* nicotinic acetylcholine receptors in brain

Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While α4β2 and α7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among α4β2*-nAChR, ABT-089 had partial agonist activity (7–23% of nicotine) and high selectivity for α4α5β2 nAChR as evidenced by loss of activity in thalamus of α5^−/− mice. ABT-089 stimulated [³H]-dopamine release (57%) exceeded the activity at α4β2* nAChR, that could be explained by the activity at α6β2* nAChR. The concentration–response relationship for ABT-089 stimulation of α6β2* nAChR was biphasic. EC₅₀ and efficacy values for ABT-089, respectively, were 28 μM and 98% at the less sensitive α6β2* nAChR and 0.11 μM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations ≤300 μM at α3β4-nAChR measured by [³H]-acetylcholine release from interpeduncular nucleus. Thus, ABT-089 is a β2* nAChR ligand with demonstrable agonist activity at α4β2* and α6β2* receptors. As one form of α6β2* nAChR is sensitive to sub-μM concentrations, we propose that this receptor in particular may contribute to the enhanced cognitive performance following low doses of ABT-089.     

Pharmacological profiles of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) on the mouse vas deferens preparation

The profiles of action of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) have been assessed in the mouse vas deferens preparation. β-FNA, but not β-CNA, demonstrated a reversible agonist action that appeared to be mediated via κ-receptor interaction. β-CNA produced an irreversible antagonism of μ-, κ- and δ-mediated agonist actions, whereas β-FNA irreversibly antagonized μ-mediated agonist effects only. This selective action of β-FNA could also be seen following administration in vivo. β-CNA and particularly β-FNA should prove valuable in the elucidation of multiple opioid receptors.     

Fibril formation by amyloid-β proteins may involve β-helical protofibrils

Abstract: We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from β-strands wound into continuous 2–3 nm-diameter β-helices. Subsequent lateral aggregation of the β-helices to form the widely observed 5–12 nm-diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated β-helix. A number of short peptide fragments of the amyloid-β (Aβ) proteins, such as Aβ34–42 [LMVGGVVIA], the nine-residue, carboxyl-terminal portion of Aβ1–42, can also form amyloid fibrils. In the present study, it was found that a β-helix formed from Aβ34–42 accounts for features suggested by published rotational resonance solid-state NMR data, including an anomalous conformation about the Gly-37–Gly-38 region and exaggerated pleating. An analogue of Aβ34–42 was synthesized in which the hydrophobic groups on the exterior of the postulated β-helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2–2.5 nm-diameter fibrils which did not associate into larger-diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from β-helical subunits.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Radiosynthesis of the α4β2 nicotinic acetylcholine receptor ligand: 5‐((1‐[11C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)vinyl)pyridine

5‐((1‐[¹¹C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)‐vinyl)pyridine ([¹¹C]‐FPVC) was synthesized from [¹¹C]‐methyl iodide and the corresponding normethyl precursor. The average time of synthesis, purification, and formulation was 42 min with an average non‐decay‐corrected radiochemical yield of 19%. The average specific radioactivity was 359 GBq/µmol (9691 mCi/µmole) at end of synthesis (EOS). Copyright © 2006 John Wiley & Sons, Ltd.     

Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in β2 subunit expression. Function was measured by on-line detection of ⁸⁶Rb⁺ efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 μM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-β-erythroidine (DHβE); and (2) stimulation with 10 μM epibatidine in the presence of 2 μM DHβE, a response that is relatively resistant to inhibition by DHβE. Deletion of the β2 subunit profoundly reduced both DHβE-sensitive and -resistant ⁸⁶Rb⁺ efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [³H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the β2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in β2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [³H]epibatidine binding was heterogeneous in that a fraction of the sites required the β2 subunit. Many remaining sites were sensitive to inhibition by -bungarotoxin indicating that a subset of the low affinity [³H]epibatidine binding are of the 7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for β2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.