Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology.
2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists.
3. The EC₅₀ of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC₅₀ for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs.
4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

     

N3,N7-diaminophenothiazinium derivatives as antagonists of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes

Derivatization of phenothiazine (PTZ, 1) has been a commonly used method to develop drugs with various pharmacological properties. In the present study, a series of PTZ derivatives 1-11 were investigated on the inhibition of the cloned α7 subunit of the human nicotinic acetylcholine receptor (α7-nAChR) expressed in Xenopus oocytes by using the two-electrode voltage-clamp technique. In the first series of experiments, the effect of unsubstituted phenothiazine 1 on α7-nAChRs was compared with that of the N3,N7-diaminophenothiazin-5-ium derivative 2, and of sequentially methylated derivatives 3-6. In the second set of experiments, the effects of N3,N7-tetra-ethyl- to n-hexylphenothiazin-5-ium derivatives 7-11 were tested. Despite the lack of activity found for 1, a reversible inhibition of α7-nAChRs, ranging from moderate to potent, was observed as a result of a sequential amine- and methylamine substitution of 1. The inhibition of ACh (100 μM)-induced currents was concentration-dependent with IC(50) values ranging from 0.4 to 16.8 μM. However, an optimal inhibitory activity was achieved by prolongation of alkyl chains up to propyl size, as found in PTZ derivative 8, whereas further lengthening of alkyl chains to n-butyl-, n-pentyl-, or n-hexyl groups resulted in inactive derivatives 9-11. The results evidently suggest the presence of a lipophilic binding pocket of narrow tolerability on the receptor protein. These results emphasize the importance of amine and/or alkylamine moieties for the inhibitory effect of PTZ derivatives and provide further insights for the development of novel antagonists targeting α7-nAChRs.     

Role of α7-nicotinic acetylcholine receptor in normal and cancer stem cells

The α7-nicotinic acetylcholine receptor (α7-nAChR) is widely known as a neurotransmitter receptor in nervous systems. α7-nAChR is also present in a variety of non-neuronal tissues, where it has been implicated in the regulation of essential cellular functions including proliferation, survival, differentiation and communication. We have recently found in breast cancer that α7-nAChR is involved in the proliferation of cancer stem cells, which constitute a minor subpopulation responsible for tumor development and metastasis. Since growing evidence suggests that α7-nAChR is present not only in mature tissues and organs but also in undifferentiated stem cells and progenitor cells, α7-nAChR emerges as a key mediator in the regulation of self-renewal and differentiation. We provide here an overview of the recent works on the expression and function of α7-nAChR in normal and cancer stem cells, and their relevance to disease-related cellular dysfunction. Understanding the role of α7-nAChR in stem cells would be of great interest for its application potential in drug discovery and in regenerative medicine.     

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit： pharmacological properties of ligand binding and function

Aim： To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors （α7 nAChRs） with those of native nAChRs containing α7 subunit （α7＊ nAChRs） in rat hippocampus and cerebral cortex. Methods： We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7＊ nAChRs in rat hippocampus and cerebral cortex. We used [125Ⅰ]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording. Results： The newly established cell line, KXα7R1, expresses homomeric α7 nAChRs that bind [125Ⅰ]-α-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kd values of native rat α7＊nAChRs from hippocampus （Kd=0.28±0.03 nmol/L） and cerebral cortex （Kd=0.33±0.05 nmol/L）. Using whole-cell current recording, the homomeric α7 nAChRs expressed in the cells were activated by acetylcholine and （-）-nicotine with EC50 values of 280±19μmol/L and 180±40μmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, a-bungarotoxin （IC50=19±2 nmol/L） and methyllycaconitine （IC50=100±10 pmol/L）. A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7＊receptors in rat brain, but it also revealed several notable differences. Conclusion： This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7＊ nAChRs.     

Development of radioligands for the imaging of α7 nicotinic acetylcholine receptors with positron emission tomography

Molecular imaging of brain structures by highly sensitive non-invasive techniques offers unique possibilities in the understanding of physiological and pathological processes in the central nervous system. In particular, the quantitative analysis by positron emission tomography (PET) of α7 nicotinic acetylcholine receptors (α7 nAChR), which are involved in different signalling pathways in the brain, is assumed to provide important information on the relation between receptor dysfunction and the pathogeneses of neuropsychiatric brain diseases, but the applicability of this imaging approach is still hampered due to insufficient imaging agents. This paper presents the recent efforts made to develop PET radiotracers targeting α7 nAChR as well as the current state of the evaluation of the most promising radiolabelled compounds in animal models and humans.     

Sonogashira coupling reaction in the synthesis of novel positive allosteric modulators of α7 nicotinic acetylcholine receptors

A series of 2-arylamino-1,3,5-triazine derivatives (4a–4g), which were designed and synthesized via Sonogashira coupling reaction, were evaluated using two-electrode voltage clamp (TEVC) recordings of human α7 nAChR expressed in Xenopus ooctyes. Compound 4g as a positive allosteric modulator (PAM) showed better efficacy than lead compound 3 (HZZ-A-11) with an EC₅₀ value of 1.23 ± 0.41 μM. Further pharmacological evaluation of compound 4g might lead to the developmental potential for therapy of cognitive deficits commonly shared by neuropsychiatric disorders, such as schizophrenia and Alzheimer’s disease.     

Role of α5* nicotinic acetylcholine receptors in the effects of acute and chronic nicotine treatment on brain reward function in mice

Objective

Allelic variation in the α5 nicotinic acetylcholine receptor (nAChR) subunit gene, CHRNA5, increases vulnerability to tobacco addiction. Here, we investigated the role of α5* nAChRs in the effects of nicotine on brain reward systems.

Materials and methods

Effects of acute (0.03125–0.5 mg/kg SC) or chronic (24 mg/kg per day; osmotic minipump) nicotine and mecamylamine-precipitated withdrawal on intracranial self-stimulation (ICSS) thresholds were assessed in wild-type and α5 nAChR subunit knockout mice. Noxious effects of nicotine were further investigated using a conditioned taste aversion procedure.

Results

Lower nicotine doses (0.03125–0.125 mg/kg) decreased ICSS thresholds in wild-type and α5 knockout mice. At higher doses (0.25–0.5 mg/kg), threshold-lowering effects of nicotine were diminished in wild-type mice, whereas nicotine lowered thresholds across all doses tested in α5 knockout mice. Nicotine (1.5 mg/kg) conditioned a taste aversion to saccharine equally in both genotypes. Mecamylamine (5 mg/kg) elevated ICSS thresholds by a similar magnitude in wild-type and α5 knockout mice prepared with minipumps delivering nicotine. Unexpectedly, mecamylamine also elevated thresholds in saline-treated α5 knockout mice.

Conclusion

α5* nAChRs are not involved in reward-enhancing effects of lower nicotine doses, the reward-inhibiting effects of nicotine withdrawal, or the general noxious effects of higher nicotine doses. Instead, α5* nAChRs regulate the reward-inhibiting effects nicotine doses that oppose the reward-facilitating effects of the drug. These data suggest that disruption of α5* nAChR signaling greatly expands the range of nicotine doses that facilitate brain reward activity, which may help explain the increased tobacco addiction vulnerability associated with CHRNA5 risk alleles.     

EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors

EVP-6124, (R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was evaluated here in vitro and in vivo. In binding and functional experiments, EVP-6124 showed selectivity for α7 nAChRs and did not activate or inhibit heteromeric α4β2 nAChRs. EVP-6124 had good brain penetration and an adequate exposure time. EVP-6124 (0.3 mg/kg, p.o.) significantly restored memory function in scopolamine-treated rats (0.1 mg/kg, i.p.) in an object recognition task (ORT). Although donepezil at 0.1 mg/kg, p.o. or EVP-6124 at 0.03 mg/kg, p.o. did not improve memory in this task, co-administration of these sub-efficacious doses fully restored memory. In a natural forgetting test, an ORT with a 24 h retention time, EVP-6124 improved memory at 0.3 mg/kg, p.o. This improvement was blocked by the selective α7 nAChR antagonist methyllycaconitine (0.3 mg/kg, i.p. or 10 μg, i.c.v.). In co-application experiments of EVP-6124 with acetylcholine, sustained exposure to EVP-6124 in functional investigations in oocytes caused desensitization at concentrations greater than 3 nM, while lower concentrations (0.3-1 nM) caused an increase in the acetylcholine-evoked response. These actions were interpreted as representing a co-agonist activity of EVP-6124 with acetylcholine on α7 nAChRs. The concentrations of EVP-6124 that resulted in physiological potentiation were consistent with the free drug concentrations in brain that improved memory performance in the ORT. These data suggest that the selective partial agonist EVP-6124 improves memory performance by potentiating the acetylcholine response of α7 nAChRs and support new therapeutic strategies for the treatment of cognitive impairment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Atypical antipsychotics as noncompetitive inhibitors of α4β2 and α7 neuronal nicotinic receptors

It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the α4β2 and α7 nicotinic receptor subtypes. The binding of [³H]-nicotine to rat cortical α4β2 receptors and [³H]-methyllycaconitine to rat hippocampal α7 receptors was not affected by any of the compounds tested. However, Rb⁺ efflux evoked either by nicotine or the selective α4β2 agonist TC-1827 from α4β2 receptors expressed in SH-EP1 cells and nicotine-evoked [³H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, α-bungarotoxin-sensitive epibatidine-evoked [³H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of α7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.     

Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The α4β2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for α4β2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the α4β2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the α4β2 receptors. Here we discuss the concept of allosterism as it pertains to the α4β2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.     

Effects of α7 nicotinic acetylcholine receptor agonists on antipsychotic efficacy in a preclinical mouse model of psychosis

Rationale  

Antipsychotics normalize responses in the DBA/2 mouse model of prepulse inhibition (PPI), a preclinical model of sensorimotor gating deficits. The α7 nicotinic acetylcholine receptor (nAChR) as a molecular target is considered an attractive approach for improvement of cognitive deficits in schizophrenia (CDS). Assessment of clinical efficacy of novel agents in CDS involves treating patients already on antipsychotic medications.     

Progress and challenges in the study of α6-containing nicotinic acetylcholine receptors

Recent progress has been made in the understanding of the anatomical distribution, composition, and physiological role of nicotinic acetylcholine receptors containing the α6 subunit. Extensive study by many researchers has indicated that a collection of α6-containing receptors representing a nicotinic sub-family is relevant in preclinical models of nicotine self-administration and locomotor activity. Due to a number of technical difficulties, the state of the art of in vitro model systems expressing α6-containing receptors has lagged behind the state of knowledge of native α6 nAChR subunit composition. Several techniques, such as the expression of chimeric and concatameric α6 subunit constructs in oocytes and mammalian cell lines have been employed to overcome these obstacles. There remains a need for other critical tools, such as selective small molecules and radioligands, to advance the field of research and to allow the discovery and development of potential therapeutics targeting α6-containing receptors for smoking cessation, Parkinson's disease and other disorders.     

Traumatic brain injury: central and peripheral role of α7 nicotinic acetylcholine receptors

Traumatic brain injury (TBI) is a significant public health concern worldwide for which there is no cure. Once trauma has occurred, multiple biochemical pathways are set into motion that leads to a chronic, neurodegenerative condition. Two of the most widely studied pathological pathways are excitotoxicity and inflammation, processes that are influenced by α7 nicotinic acetylcholine receptors (nAChR). Previous studies have found a bilateral decrease in α7 nAChR expression in regions of the cortex and hippocampus that occurs in relation to injury severity. Subsequent studies showed that this decrease was evident in some parts of the hippocampus as early as 1 hour post-injury and remained decreased through 21 days. Other ligand-gated ion channels, such as non-α7 nAChRs and n-methyl-D-aspartate (NMDA) receptors did not show a similar widespread and consistent pattern of change following TBI, nor did the G-protein coupled muscarinic acetylcholine receptors, suggesting that the α7 nAChR could be a key mediator in the pathophysiology of traumatic brain injury. In addition to its expression in the brain, the α7 nAChR has been found outside of the central nervous system (CNS) on many different cell types, including peripheral blood leukocytes, where they have a role in the cholinergic antiinflammatory pathway, and have recently been identified on platelets where they may have a role in activation. How these receptors are regulated in response to injury has not been investigated, but could potentially serve as a marker of neurodegeneration as has been done in Alzheimer's disease and schizophrenia. In this review, we will detail the role of α7 nAChR following TBI as well as explore the evidence of this receptor subtype in regards to blood component (leukocytes and platelets) involvement and the potential influence TBI has on peripheral expression and function.     

Activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration in mice

Cholinergic anti-inflammatory actions have been shown to result mainly from the activation of α7 nicotinic acetylcholine receptors. Here, we investigated the possible role of α7 nicotinic acetylcholine receptors in the pathogenesis of indomethacin-induced small intestinal ulceration in mice. Male C57BL/6 mice were given indomethacin (10 mg/kg, s.c.), and sacrificed 24 h later. Nicotine (0.3–3 mg/kg) and PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors; 1–10 mg/kg) were administered i.p. twice, at 0.5 h before and 8 h after indomethacin treatment, while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors; 10 mg/kg was administered twice, at 0.5 h before each nicotine treatment. Indomethacin caused severe hemorrhagic lesions in the small intestine with marked increases in myeloperoxidase (MPO) activity and inducible nitric oxide synthase (iNOS) expression in the mucosa. Pretreatment with nicotine reduced the severity of intestinal lesions in a dose-dependent manner. The protective effect of nicotine was mimicked by PNU-282987 and significantly attenuated by methyllycaconitine. The increases in MPO activity and iNOS expression induced by indomethacin were also significantly suppressed by nicotine and PNU-282987. Immunohistochemical study showed that the expression of α7 nicotinic acetylcholine receptors was clearly enhanced in the submucosa of the damaged area following indomethacin treatment. These results suggest that the activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration, and that this effect may result from the inhibition of iNOS expression and neutrophil migration.     

Role of channel activation in cognitive enhancement mediated by α7 nicotinic acetylcholine receptors

Background and purpose:

Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.

Experimental approach:

Two structurally related α7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm.

Key results:

NS6784 activated human and rat α7 nAChR with EC₅₀s of 0.72 and 0.88 µM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at α7 nAChR (<2% in oocytes, ≤8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of α7 nAChRs or using the slowly desensitizing α7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941.

Conclusions and implications:

Collectively, these findings suggest that a degree of α7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to α7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of α7 nAChR antagonists with favourable CNS penetration.     

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents

Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents (I_ACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I_ACh in concentration-dependent and reversible manners, and the blocking effect on I_ACh was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC₅₀) of quercetin was 45.4 ± 10.1 μM. Quercetin-mediated I_ACh inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca²⁺, the action of quercetin was independent of extracellular Ca²⁺ concentration, indicating that the presence of extracellular Ca²⁺ might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.     

Stress and the α7 nicotinic acetylcholine receptor

Nicotine is well known for its deleterious effects on human health, and it has long been known that nicotine interacts with the stress axis in both man and in laboratory animals. Nicotine also has beneficial effects upon cognition, and an emerging literature has demonstrated that it may play a protective or palliative role in diseases such as Alzheimer's disease and schizophrenia. Recent advances have permitted scientists to identify the specific subtypes of nicotinic receptors responsible for the drugs varied physiological effects. The α7 subunit of the nicotinic acetylcholine receptor (NAchRα7), has been identified as a significant mediator of nicotine's interactions with the stress axis and human disease. The NAchRα7 has also been shown to have neuroprotective effects via multiple pathways, making it a logical target for the treatment of a number of brain disorders.