Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drug modulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium may alter this balance and modulate the ability of PNU to potentiate α7.

The alpha 7 nicotinic acetylcholine receptor agonist PHA 568487 dampens inflammation in PBMCs from patients with newly discovered coronary artery disease.

The alpha 7 nicotinic acetylcholine receptor (α7nAChR) regulates inflammation in experimental models and is expressed in human peripheral blood mononuclear cells (PBMCs) and in human atherosclerotic plaques. However, its role in regulating inflammation in patients with cardiovascular disease is unknown. This study aims to investigate whether α7nAChR stimulation can reduce the inflammatory response in PBMCs from patients with newly diagnosed coronary artery disease (CAD). Human PBMCs, extracted from patients with verified CAD (n = 38) and control participants with healthy vessels (n = 38), were challenged in vitro with lipopolysaccharide (LPS) in combination with the α7nAChR agonist PHA 568487. Cytokine levels of the supernatants were analyzed using a multiplex immunoassay. Patients in the CAD group were reexamined after 6 mo. The immune response to LPS did not differ between PBMCs from control and CAD groups. α7nAChR stimulation decreased TNFα in both control and CAD groups. The most pronounced effect of α7nAChR stimulation was observed in patients with CAD at their first visit, where 15 of 17 cytokines were decreased [IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-17A, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1ß, and TNFα]. In conclusion, stimulation with α7nAChR agonist PHA 568487 dampens the inflammatory response in human PBMCs. This finding suggests that the anti-inflammatory properties of the α7nAChR may have a role in treating CAD.NEW & NOTEWORTHY The α7nAChR is an important regulator of inflammation; however, its anti-inflammatory function in patients with newly diagnosed coronary artery disease (CAD) remains unclear. We demonstrate that stimulation of α7nAChR with PHA 568487 attenuates the inflammatory response in immune cells extracted from healthy controls and patients with newly diagnosed CAD, with a more pronounced effect observed in patients with CAD. This suggests that the anti-inflammatory properties of α7nAChR may have a role in treating chronic inflammatory diseases.

Activation of the Alpha 7 Nicotinic Acetylcholine Receptor by GTS-21 Mitigates Contrast Nephropathy in a Rat Model.

INTRODUCTION: Contrast nephropathy (CN) is characterized by oxidative stress, vasoconstriction, tubular toxicity, and hypoxia of the renal medulla. We aimed to test the therapeutic effects of an α7 nicotinic acetylcholine receptor (nAChR) agonist, GTS-21, in an experimental CN model. METHODS: Male Sprague-Dawley rats (n = 40) were divided into 4 groups: saline-treated control, GTS-21-treated control, contrast, and GTS-21-treated contrast groups. Starting on the 1st day, GTS-21 (4 mg/kg, intraperitoneally) or saline was administered twice a day for 3 days. CN was induced on the second day by intravenous injection of indomethacin (10 mg/kg), l-NAME (10 mg/kg), and a contrast agent with high osmolarity (6 mL/kg; Urografin 76%). At the 72nd hour, blood and kidney samples were obtained for the determination of biochemical, histological, and gene expression parameters. RESULTS: Compared to those in control rats, the elevated serum BUN level in the contrast group decreased with GTS-21 treatment, while H&E staining and TUNEL assays showed that contrast-induced renal injury was improved by GTS-21. Moreover, GTS-21 treatment in the CN also increased the antioxidant glutathione level. In the contrast group, a significant increase in IL-6 expression and a decrease in TGF-ß expression were observed; however, GTS-21 treatment decreased IL-6 expression and increased TGF-ß expression. CONCLUSION: GTS-21 significantly alleviated renal injury parameters through antioxidant, anti-inflammatory, and antiapoptotic mechanisms in the CN model.

Alpha 7 Nicotinic Acetylcholine Receptor Agonist PHA 568487 Reduces Acute Inflammation but Does Not Affect Cardiac Function or Myocardial Infarct Size in the Permanent Occlusion Model.

Stimulation of the alpha 7 nicotinic acetylcholine receptor (α7nAChR) has shown beneficial effects in several acute inflammatory disease models. This study aims to examine whether treatment with the selective α7nAChR agonist PHA 568487 can dampen inflammation and thereby improve cardiac function after myocardial infarction in mice. The possible anti-inflammatory properties of α7nAChR agonist PHA 568487 were tested in vivo using the air pouch model and in a permanent occlusion model of acute myocardial infarction in mice. Hematologic parameters and cytokine levels were determined. Infarct size and cardiac function were assessed via echocardiography 24 h and one week after the infarction. Treatment with α7nAChR agonist PHA 568487 decreased 12 (CCL27, CXCL5, IL6, CXCL10, CXCL11, CXCL1, CCL2, MIP1a, MIP2, CXCL16, CXCL12 and CCL25) out of 33 cytokines in the air pouch model of acute inflammation. However, α7nAChR agonist PHA 568487 did not alter infarct size, ejection fraction, cardiac output or stroke volume at 24 h or at 7 days after the myocardial infarction compared with control mice. In conclusion, despite promising immunomodulatory effects in the acute inflammatory air pouch model, α7nAChR agonist PHA 568487 did not affect infarct size or cardiac function after a permanent occlusion model of acute myocardial infarction in mice. Consequently, this study does not strengthen the hypothesis that stimulation of the α7nAChR is a future treatment strategy for acute myocardial infarction when reperfusion is lacking. However, whether other agonists of the α7nAChR can have different effects remains to be investigated.

Silent agonists for α7 nicotinic acetylcholine receptors.

We discuss models for the activation and desensitization of α7 nicotinic acetylcholine receptors (nAChRs) and the effects of efficacious type II positive allosteric modulators (PAMs) that destabilize α7 desensitized states. Type II PAMs such as PNU-120596 can be used to distinguish inactive compounds from silent agonists, compounds that produce little or no channel activation but stabilize the non-conducting conformations associated with desensitization. We discuss the effects of α7 nAChRs in cells of the immune system and their roles in modulating inflammation and pain through what has come to be known as the cholinergic anti-inflammatory system (CAS). Cells controlling CAS do not generate ion channel currents but rather respond to α7 drugs by modulating intracellular signaling pathways analogous to the effects of metabotropic receptors. Metabotropic signaling by α7 receptors appears to be mediated by receptors in nonconducting conformations and can be accomplished by silent agonists. We discuss electrophysiological structure-activity relationships for α7 silent agonists and their use in cell-based and in vivo assays for CAS regulation. We discuss the strongly desensitizing partial agonist GTS-21 and its effectiveness in modulation of CAS. We also review the properties of the silent agonist NS6740, which is remarkably effective at maintaining α7 receptors in PAM-sensitive desensitized states. Most silent agonists bind to sites overlapping those for orthosteric agonists, but some appear to bind to allosteric sites. Finally, we discuss α9* nAChRs and their potential role in CAS, and ligands that will be useful in defining and distinguishing the specific roles of α7 and α9 in CAS.

Potentiation and allosteric agonist activation of α7 nicotinic acetylcholine receptors: binding sites and hypotheses.

Considerable progress has been made in recent years towards the identification and characterisation of novel subtype-selective modulators of nicotinic acetylcholine receptors (nAChRs). In particular, this has focussed on modulators of α7 nAChRs, a nAChR subtype that has been identified as a target for drug discovery in connection with a range of potential therapeutic applications. This review focusses upon α7-selective modulators that bind to receptor sites other than the extracellular 'orthosteric' agonist binding site for the endogenous agonist acetylcholine (ACh). Such compounds include those that are able to potentiate responses evoked by orthosteric agonists such as ACh (positive allosteric modulators; PAMs) and those that are able to activate α7 nAChRs by direct allosteric activation in the absence of an orthosteric agonist (allosteric agonists or 'ago-PAMs'). There has been considerable debate about the mechanism of action of α7-selective PAMs and allosteric agonists, much of which has centred around identifying the location of their binding sites on α7 nAChRs. Based on a variety of experimental evidence, including recent structural data, there is now clear evidence indicating that at least some α7-selective PAMs bind to an inter-subunit site located in the transmembrane domain. In contrast, there are differing hypotheses about the site or sites at which allosteric agonists bind to α7 nAChRs. It will be argued that the available evidence supports the conclusion that direct allosteric activation by allosteric agonists/ago-PAMs occurs via the same inter-subunit transmembrane site that has been identified for several α7-selective PAMs.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

New Synthetic Caffeine Analogs as Modulators of the Cholinergic System.

Alzheimer's disease is a multifactorial neurodegenerative disorder. Since cholinergic deficit is a major factor in this disease, two molecular targets for its treatment are the acetylcholinesterase (AChE) and the nicotinic acetylcholine receptors (nAChRs). Given that caffeine is a natural compound that behaves as an AChE inhibitor and as a partial agonist of nAChRs, the aim of this work was to synthetize more potent bifunctional caffeine analogs that modulate these two molecular targets. To this end, a theophylline structure was connected to a pyrrolidine structure through a methylene chain of different lengths (3 to 7 carbon atoms) to give compounds 7-11 All caffeine derivatives inhibited the AChE, of which compound 11 showed the strongest effect. Electrophysiological studies showed that all compounds behave as agonists of the muscle and the neuronal α7 nAChR with greater potency than caffeine. To explore whether the different analogs could affect the nAChR conformational state, the nAChR conformational-sensitive probe crystal violet (CrV) was used. Compounds 9 and 10 conduced the nAChR to a different conformational state comparable with a control nAChR desensitized state. Finally, molecular docking experiments showed that all derivatives interacted with both the catalytic and anionic sites of AChE and with the orthosteric binding site of the nAChR. Thus, the new synthetized compounds can inhibit the AChE and activate muscle and α7 nAChRs with greater potency than caffeine, which suggests that they could be useful leaders for the development of new therapies for the treatment of different neurologic diseases. SIGNIFICANCE STATEMENT: In this work we synthetized caffeine derivatives which can inhibit acetylcholinesterase and activate both muscle and α7 nicotinic acetylcholine receptors (nAChRs) with higher potency than caffeine. These analogs can be divided into two groups: a non-desensitizing and a desensitizing nAChR group. From the nAChR non-desensitizing group, we propose compound 11 as the most interesting analog for further studies since it inhibits acetylcholinesterase with the highest potency and activates the nAChRs in the picomolar range without inducing receptor desensitization.

α7 nicotinic acetylcholine receptor agonist GTS-21 attenuates DSS-induced intestinal colitis by improving intestinal mucosal barrier function.

BACKGROUND AND AIMS: Cholinergic output, which could modulate innate immune responses through stimulation of α7 nicotinic acetylcholine receptor (α7nAChR), might be a target to minimize tissue damage in autoimmune disease. GTS-21, a selective α7nAChR agonist, has previously demonstrated to inhibit synovium inflammation in rheumatoid arthritis. In this study, we investigated the effect of GTS-21 on dextran sulfate sodium (DSS)-induced colitis model and its potential mechanism. METHODS: Male BABL/c mice (n = 32) were randomly divided into four groups: normal control group, DSS-induced colitis group, GTS-21 treatment with or without α7nAChR antagonist α-BGT treatment group. Disease activity index (DAI), histological activity index (HAI) and colonic macroscopic damage were evaluated. Fluorescein isothiocyanate (FITC)-dextran assay was applied to measure intestinal permeability. The expressions of tight junction (TJ) proteins and NF-κB associated proteins were detected by Western blot. RESULTS: GTS-21 could decrease DAI scores, HAI scores, intestinal permeability and reduce the intestinal bacterial translocation in DSS-induced colitis group, whereas α7nAChR antagonist α-BGT could impair this protective influence. The expressions of TJ proteins were increased with administration of GTS-21 both in vivo and in vitro. Furthermore, GTS-21 also inhibited the NF-қB activation in intestinal epithelial cells and colitis model, while α-BGT reversed the inhibitory effect. CONCLUSION: The α7nAChR agonist GTS-21 attenuated DSS-induced colitis through increasing expressions of TJ proteins in colon tissues and improved intestinal barrier function, which might be due to  modulating NF-қB activation in intestinal epithelial cells.

Celiac vagus nerve mediates expression of the acetylcholine receptor α7nAChR on monocytes in the spleen.

The spleen is required for the vagal cholinergic anti-inflammatory activity to maintain systemic immune homeostasis, but the underlying mechanism of this function is not fully understood yet. We hypothesized that vagus nerve mediates alpha 7 nicotinic acetylcholine receptor (α7nAChR) expression in monocytes, an essential regulator of cholinergic anti-inflammatory activity, and the spleen is essential site for this process. To verify this hypothesis, mice were subjected to splenectomy or celiac vagotomy. The level of α7nAChR expression in circulating monocytes was analyzed by real-time PCR. Impact of α7nAChR agonist PNU282987 on LPS-evoked release of TNF-α and IL-1ß from circulating monocytes was assessed by ELISA. The effect of norepinephrine (NE), acetylcholine (ACh) and neuregulin-1 (NRG-1) on α7nAChR expression was detected by real-time PCR. We found that splenectomy or celiac vagotomy abrogated α7nAChR expression in circulating monocytes. LPS-induced release of TNF-α and IL-1ß from these monocytes was not alleviated significantly by PNU282987 as compared with that of sham mice. NE and ACh addition fails to stimulate α7nAChR expression, but, NRG-1 treatment can significantly induce α7nAChR expression in these monocytes compared with untreated cells in vitro. Overall, our results reveal that celiac vagus nerve mediates α7nAChR expression in monocytes, and the spleen is indispensable site for this process.

Differential Activation and Desensitization States Promoted by Noncanonical α7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential α7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human α7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of α7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.

Evidence for Alpha7 Nicotinic Receptor Activation During the Cough Suppressing Effects Induced by Nicotine and Identification of ATA-101 as a Potential Novel Therapy for the Treatment of Chronic Cough.

Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α 4 ß 2)-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α 7-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α 7) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α 7 nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α 7 nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α 7 nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α7 nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α7 nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.

Expression of the α7 Nicotinic Acetylcholine Receptor Is Critically Required for the Antifibrotic Effect of PHA-543613 on Skin Fibrosis.

INTRODUCTION: Targeting the α7 nicotinic acetylcholine receptor (α7nAChR) has recently been suggested as a potential new treatment for fibrotic skin diseases. Here, we performed a genetic and pharmacologic approach to clarify the role of this receptor in the bleomycin (BLM) mouse model of skin fibrosis using α7nAChR KO mice. METHODS: We analyzed the expression of extracellular matrix (ECM) components in murine skin using quantitative RT-PCR, pepsin digestion/SDS-PAGE of proteins and performed hydroxyproline assays as well as histological/immunohistochemical staining of skin sections. To identity the target cells of the α7nAChR agonist PHA-543613, we used murine dermal fibroblasts (MDF). We tested their response to the profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) and utilized gene silencing to elucidate the role of the α7nAChR. RESULTS: We confirmed our previous findings on C3H/HeJ mice and detected a suppressive effect of PHA-543613 on BLM-induced skin fibrosis in the mouse strain C57BL/6J. This antifibrotic effect of PHA-543613 was abrogated in α7nAChR-KO mice. Interestingly, α7nAChR-KO animals exhibited a basal profibrotic signature by higher RNA expression of ECM genes and hydroxyproline content than WT mice. In WT MDF, PHA-543613 suppressed ECM gene expression induced by TGF-ß1. Gene silencing of α7nAChR by small interfering RNA neutralized the effects of PHA-543613 on TGF-ß1-mediated ECM gene expression. CONCLUSION: In summary, we have identified the α7nAChR as the essential mediator of the antifibrotic effect of PHA-543613. MDF are directly targeted by PHA-543613 to suppress collagen synthesis. Our findings emphasize therapeutic exploitation of α7nAChR receptor agonists in fibrotic skin diseases.

The alpha-7 nicotinic acetylcholine receptor agonist GTS-21 engages the glucagon-like peptide-1 incretin hormone axis to lower levels of blood glucose in db/db mice.

AIM: To establish if alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a blood glucose-lowering action in db/db mice, and to test if this action requires coordinate α7nAChR and GLP-1 receptor (GLP-1R) stimulation by GTS-21 and endogenous GLP-1, respectively. MATERIALS AND METHODS: Blood glucose levels were measured during an oral glucose tolerance test (OGTT) using db/db mice administered intraperitoneal GTS-21. Plasma GLP-1, peptide tyrosine tyrosine 1-36 (PYY1-36), glucose-dependent insulinotropic peptide (GIP), glucagon, and insulin levels were measured by ELISA. A GLP-1R-mediated action of GTS-21 that is secondary to α7nAChR stimulation was evaluated using α7nAChR and GLP-1R knockout (KO) mice, or by co-administration of GTS-21 with the dipeptidyl peptidase-4 inhibitor, sitagliptin, or the GLP-1R antagonist, exendin (9-39). Insulin sensitivity was assessed in an insulin tolerance test. RESULTS: Single or multiple dose GTS-21 (0.5-8.0 mg/kg) acted in a dose-dependent manner to lower levels of blood glucose in the OGTT using 10-14 week-old male and female db/db mice. This action of GTS-21 was reproduced by the α7nAChR agonist, PNU-282987, was enhanced by sitagliptin, was counteracted by exendin (9-39), and was absent in α7nAChR and GLP-1R KO mice. Plasma GLP-1, PYY1-36, GIP, glucagon, and insulin levels increased in response to GTS-21, but insulin sensitivity, body weight, and food intake were unchanged. CONCLUSIONS: α7nAChR agonists improve oral glucose tolerance in db/db mice. This action is contingent to coordinate α7nAChR and GLP-1R stimulation. Thus α7nAChR agonists administered in combination with sitagliptin might serve as a new treatment for type 2 diabetes.

Activation of α7 Nicotinic Acetylcholine Receptor by its Selective Agonist Improved Learning and Memory of Amyloid Precursor Protein/Presenilin 1 (APP/PS1) Mice via the Nrf2/HO-1 Pathway.

BACKGROUND To reveal the mechanism underlying the effect of alpha7 nicotinic acetylcholine receptor (nAChR) on neurodegeneration in Alzheimer disease (AD), the influence of the receptor on recognition in APP/PS1 mice was evaluated by using its selective agonist (PNU-282987). MATERIAL AND METHODS APP/PS1 and wild-type (WT) mice were treated with PNU or saline, respectively, for 7 days at the ages of 6 and 10 months. RESULTS Morris water maze analysis showed that both at 6 and 10 months of age, PNU treatment enhanced the learning and memory of APP/PS1 mice. However, PNU treatment did not alter the number of senile plaques. Furthermore, a higher protein expression of Nrf2/HO-1, ADAM10, SYP, and SNAP-25, and a lower level of oxidative stress, were observed in the hippocampus of APP/PS1 mice treated with PNU compared with the control group. CONCLUSIONS The results indicated that the activation of alpha7 nAChR by PNU improved the learning and memory of mice carrying the APP/PS1 mutation, regulated the levels of enzymes that mediate APP metabolization to reduce ß-amyloid peptide damage, and decreased the level of oxidative stress and maintained synaptic plasticity, in which the mechanism might be enhancement of the Nrf2/HO-1 pathway.

Roles of alpha-7 nicotinic acetylcholine receptors and spleen in the lung injury induced by a repeated saline lavage in rat.

BACKGROUND: The study aimed to determine whether or notα7 nicotinic acetylcholine receptors (α7nAChR) induce anti-inflammatory effects directly in the lung or through the spleen pathway in a sterile model of lung injury by saline lavage. METHODS: Male Sprague Dawley rats were divided into seven groups; Sham, splenectomy (SPX), saline lavage (LAV), LAV treated with α7nAChR agonist nicotine (LAV + NIC), and LAV treated with NIC and a selective α7nAChR antagonist MLA (LAV+MLA+NIC), LAV and splenectomy (LAV+SPX), and LAV+SPX treated with nicotine (LAV+SPX+NIC). Tracheostomy and catheterization of the femoral artery were performed under deep anesthesia. Animals were subjected to volume-controlled ventilation and lung injury by 10 repeated saline lavages. Splenectomy was achieved one week before the induction of lung injury. The recovery phase lasted for 3 h, and drugs were injected 1 h after the last lavage. RESULTS: Mean arterial blood pressure (MBP), heart rate (HR), PaO2, PaO2/FiO2 ratio, and pH decreased, whereas, maximal inspiratory (MIP) and expiratory (MEP) pressures, and PaCO2 increased 1 h after the saline lavage. Nicotine corrected entirely all the above parameters in the LAV + NIC group. MLA or SPX prevented the effects of nicotine on the above parameters, except that MLA had no extra effect on MIP or MEP. In addition, nicotine improved lung compliance in the LAV + NIC and LAV + SPX + NIC groups, though it was inhibited by MLA in the LAV + MLA + NIC group. The increases of plasma and lung tissue malondialdehyde (MDA) in the LAV group were diminished by nicotine, whereas, MLA and SPX prevented these reductions. Besides, nicotine could reduce plasma MDA in the LAV + SPX + NIC group. Total BAL cell count, protein BAL/protein plasma ratio, and lung histological scores were attenuated by nicotine in the LAV + NIC group, whereas, MLA reversed the mentioned alterations in the LAV + MLA + NIC group. However, splenectomy could not stop the decreasing effect of nicotine on the total BAL cell in the LAV + SPX + NIC group. CONCLUSIONS: In this study, we indicated that α7nAChR and spleen play roles in cholinergic anti-inflammatory pathways in saline lavage-induced lung injury. However, our results are in favor of at least some direct effects of α 7nAChR in the lung.

Inflammation Regulation via an Agonist and Antagonists of α7 Nicotinic Acetylcholine Receptors in RAW264.7 Macrophages.

The α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems and is closely related to a variety of nervous system diseases and inflammatory responses. The α7 nAChR subtype plays a vital role in the cholinergic anti-inflammatory pathway. In vivo, ACh released from nerve endings stimulates α7 nAChR on macrophages to regulate the NF-κB and JAK2/STAT3 signaling pathways, thereby inhibiting the production and release of downstream proinflammatory cytokines and chemokines. Despite a considerable level of recent research on α7 nAChR-mediated immune responses, much is still unknown. In this study, we used an agonist (PNU282987) and antagonists (MLA and α-conotoxin [A10L]PnIA) of α7 nAChR as pharmacological tools to identify the molecular mechanism of the α7 nAChR-mediated cholinergic anti-inflammatory pathway in RAW264.7 mouse macrophages. The results of quantitative PCR, ELISAs, and transcriptome analysis were combined to clarify the function of α7 nAChR regulation in the inflammatory response. Our findings indicate that the agonist PNU282987 significantly reduced the expression of the IL-6 gene and protein in inflammatory macrophages to attenuate the inflammatory response, but the antagonists MLA and α-conotoxin [A10L]PnIA had the opposite effects. Neither the agonist nor antagonists of α7 nAChR changed the expression level of the α7 nAChR subunit gene; they only regulated receptor function. This study provides a reference and scientific basis for the discovery of novel α7 nAChR agonists and their anti-inflammatory applications in the future.

Activation of α-7 Nicotinic Acetylcholine Receptor Attenuates Cardiac Inflammation Through NLRP3/Caspase-1/IL-18 Pathway.

Activation of α-7 nicotinic acetylcholine receptor (α7nAChR) receptor might induce cardiac inflammation, cardiac remodeling, and dysfunction. In this regard, this study aims to clarify the role and mechanism of α7nAChR in the process of cardiac inflammation and damage. Normal male C57BL/6J and NLRP3-knockout mice were used to evaluate the effect of PHA-543613, a selective agonist of α7nAChR, on cardiac inflammation and possible involvement of NLRP3/Caspase-1/IL-18 using western blotting and ELISA. Activation of α7nAChR using PHA-543613 (NE), at the doses of 0.5 mg/kg and 1 mg/kg, induced cardiac inflammation. In addition, both in vivo and in vitro studies showed higher expression of NLRP3 and higher activation of Caspase-1 and IL-18 after treating animals with NE. On the other hand, we did not observe any significant changes in inflammatory cytokines and cardiac inflammation after administration of NE in NLRP3-knockout mice. It could be concluded that blocking the NLRP3/Caspase-1/IL-18 pathway can simultaneously inhibit the inflammatory response mediated by α7nAChR and it would a novel target for inhibiting cardiac inflammation and remodeling.

Suppression of neuroinflammation by an allosteric agonist and positive allosteric modulator of the α7 nicotinic acetylcholine receptor GAT107.

BACKGROUND: The α7 nicotinic acetylcholine receptor (α7 nAChR) negatively regulates the synthesis and release of pro-inflammatory cytokines by immune cells. Our previous studies showed that in encephalitogenic T cells, α7 nAChR expression is upregulated and that activation of the cholinergic system can attenuate experimental autoimmune encephalomyelitis (EAE). GAT107 is an allosteric agonist and positive allosteric modulator (ago-PAM) of α7 nAChR that can produce persistent activation of this receptor. Therefore, in the present study, we investigated the effect of GAT107 on neuroinflammation in EAE, the animal model used for the study of multiple sclerosis (MS) via α7 nAChR, and the inflammatory pathways involved. METHODS: EAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG35-55) in C57BL/6 mice. EAE mice were treated with the ago-PAM GAT107 or a placebo for 9 days, starting from the day of EAE induction. Clinical assessment and immunological evaluation of immune cells and cytokine production was performed. RESULTS: Following activation of the α7 nAChR by GAT107 during EAE, disease severity was significantly reduced by 70% and was correlated with a reduction in the extent of neuroinflammation in the CNS. The treatment reduced encephalitogenic T cell proliferation and the production of pro-inflammatory cytokines, as well as increased the production of the anti-inflammatory cytokine IL-10. Furthermore, the expression of immune cell markers was altered by GAT107 treatment, which induced a significant reduction in macrophages, dendritic cells, and B cells, as well as a reduction in anti-MOG35-55 antibodies. Additionally, GAT107 was found to directly activate α7 nAChR in murine macrophage RAW264.7 cells and in human PBMCs derived from MS patients and healthy donors. CONCLUSIONS: Our results show that GAT107 can be a useful molecule for harnessing the cholinergic anti-inflammatory pathway for long-lasting and wide-ranging modulation and downregulation of neuroinflammation in EAE.

Natural Polyhydroxy Flavonoids, Curcuminoids, and Synthetic Curcumin Analogs as α7 nAChRs Positive Allosteric Modulators.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain, and inflammation. Allosteric modulation of this receptor might be advantageous to reduce the toxicity in comparison with full agonists. Our previous results obtained with some hydroxy-chalcones, which were identified as positive allosteric modulators (PAMs) of α7 nAChR, prompted us to evaluate the potential of some structurally related naturally occurring flavonoids and curcuminoids and some synthetic curcumin analogues, with the aim of identifying new allosteric modulators of the α7 nAChR. Biological evaluation showed that phloretin, demethoxycurcumin, and bis-demethoxicurcuming behave as PAMs of α7 nAChR. In addition, some new curcumin derivatives were able to enhance the signal evoked by ACh; the activity values found for the tetrahydrocurcuminoid analog 23 were especially promising.