Dual Effects of Acetylcholine on Mast Cell-Nerve Interactions.

Nicotinic and muscarinic cholinergic receptor binding in the human hippocampal formation during development and aging

Is binding to nicotinic acetylcholine and dopamine receptors related to working memory in rats?

Free cytotransmitter acetylcholine (ACh) is present in physiologically relevant concentrations in human skin, and ACh and cholinergic drugs alter vital functions of keratinocytes (KCs). KCs respond to ACh via classical ACh receptor types that use Ca2+ as a second messenger. The repertoire of cholinergic receptors changes with cell maturation, so that at each stage of their development, KCs respond to ACh via different combinations of nicotinic (nAChR) and muscarinic (mAChR) receptors. Basal KCs respond to ACh predominantly via α3β2(β4) ± α5 nAChRs and the M3 mAChR; prickle KCs have more α5‐containing α3 nAChRs and also express α9 nAChR as well as M4 and M5 mAChRs; granular KCs posses mainly α7 nAChR and M1 mAChR. We used three independent approaches to elucidate role of each receptor in KC biology: 1) Subtype selective drugs, 2) Gene silencing with small interfering RNA or antisense oligonucleotides, and 3) Gene knockout in transgenic mice. Obtained results indicate that KC nAChRs and mAChR exhibit synergistic control of cell adhesion and crawling locomotion. KC cell‐cell adhesion is regulated through α3 and α9 nAChRs as well as M3 mAChR. KC chemokinesis is controlled by α3 and α7 nAChRs and M3 and M4 mAChRs. KC chemotaxis toward nicotinic agonists is mediated by α7 nAChR. These findings offer novel insights into the mechanisms of ACh‐mediated modulation of epidermalization and may aid the development of novel methods to promote wound healing and inhibit tumor metastasis.

Introduction Steatosis is a known risk factor in hepatic surgery and it is associated with postoperative morbidity and mortality. Acetylcholine (ACh) is the major neurotransmitter of vagus nerve and it is released as a response to stressful events like trauma or infection. Stress can also activate the HPA axis that drives the production of cortisol. Both cortisol and ACh have been proved beneficial in experimental models of sepsis, liver transplantation or myocardial ischemia, among other pathologies. In this study, we will study the relationship between cortisol and ACh and their effects on regeneration and injury in seatotic livers submitted to partial hepatectomy (PH) under vascular occlusion, a common technique used in hepatic surgery to avoid bleeding. Materials & Methods We determined hepatic levels of ACh and its receptors in obese Zucker rats that underwent partial hepatectomy under vascular occlusion. The ACTH-cortisol pathway was pharmacologically modulated and the effects on hepatic injury and regeneration, as well as its underlying mechanisms of action, were evaluated. Results There is a significant ACh accumulation driven by the administration of cortisol in steatotic livers submitted to PH under vascular occlusion, that consequently leads to an overexpression of a7 nicotinic and M3 muscarinic ACh receptors. However, administration of ACh prior to surgery increases injury and impairs regeneration via M3 mACh receptor. Interestingly, M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, did confer eprotection against liver damage caused by the surgical procedure and enhanced regeneration. Moreover, administration of ACh reduced PI3K/Akt hepatic expression and consequently reduced levels of markers of oxidative stress, neutrophil accumulation and edema. In consonance with the results presented above, treatment with an M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, reduced the hepatic levels of these harmful markers as a result of an overexpression of PI3K and pAKT in these steatotic livers. Thus, this indicates a lesser role of a7 nAChR. Conclusions The reported benefits of cortisol in PH cannot be extrapolated in situations of PH under vascular occlusion in steatotic livers. Cortisol is responsible of the accumulation of acetylcholine in these livers submitted to partial hepatectomy with vascular occlusion. ACh exerts its action through the M3 ACh receptor, as it exacerbates liver damage and oxidative stress and impairs regeneration. Strategies aimed to block cortisol activity in order to block ACh accumulation and/or blockade of M3 mAChR are probably worthwhile to protect steatotic livers and reduce the incidence of postoperative complications following PH under vascular occlusion.

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.

Irritable bowel syndrome (IBS) is a chronic functional bowel disorder characterized by intestinal dysmotility. Changes in intestinal microbiota (dysbiosis) can lead to alterations in neuro-muscular functions in the gut. Toll-like receptors (TLRs) 2 and 4 recognize intestinal bacteria and are involved in the motor response induced by gastrointestinal (GI) neurotransmitters. Acetylcholine (ACh) is a well-known neurotransmitter involved in the regulation of GI motility. This study aimed to evaluate the role of TLR2 and TLR4 in the intestinal motor-response induced by ACh in the mouse ileum, as well as the expression and function of the muscarinic and nicotinic ACh receptors. Muscle contractility studies showed that the contractions induced by ACh were significantly lower in TLR2−/− and TLR4−/− with respect to WT mice. In WT mice, the contractions induced by ACh were reduced in the presence of AF-DX AF-DX 116 (a muscarinic ACh receptor (mAChR) M2 antagonist), 4-DAMP (a mAChR M3 antagonist), mecamylamine (a nicotinic AChR receptor (nAChR) α3β4 antagonist) and α-bungarotoxin (a nAChR α7 antagonist). In TLR2−/− mice, the contractions induced by ACh were increased by AF-DX 116 and mecamylamine. In TLR4−/− mice, the contractions induced by ACh were reduced by α-bungarotoxin and 4-DAMP. The mRNA and protein expressions of M3 and α3 receptors were diminished in the ileum from TLR2−/− and TLR4−/− with respect to WT mice. However, the levels of mRNA and protein of β4 were diminished only in TLR4−/− but not in TLR2−/− mice. In conclusion, our results show that TLR2 and TLR4 modulates the motor responses to ACh in the mouse ileum. TLR2 acts on muscarinic M2 and M3 and nicotinic α3β4 ACh receptors, while TLR4 acts on muscarinic M3 and nicotinic α3β4 and α7 ACh receptors.

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of alpha7 and 5-HT3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of alpha7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the alpha7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.

Traumatic brain injury (TBI) poses a significant socioeconomic burden in the world. The long lasting consequences in cognitive impairments are often underreported and its mechanisms are unclear. In this perspective, cholinergic dysfunction and therapeutic strategy targeting this will be reviewed. Novel agents that can target specific subtype of acetylcholine receptors have been developed over the recent years and are at various stages of development, which include AR-R 17779, GTS-21, SSR-180711A, AR-{"type":"entrez-nucleotide","attrs":{"text":"R17779","term_id":"771389","term_text":"R17779"}}R17779, and PNU-282987. A detailed review on this topic has been previously published (Shin and Dixon, 2015).

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.

In the brain, alpha7 neuronal nicotinic acetylcholine receptors (α7 nAChRs) have a special role amongst nAChRs. α7 nAChRs are forming homopentamers, they display a high permeability for Ca2+, and they are the most prevalent nAChRs in the brain. α7 nAChRs are found at the highest concentration in the hippocampus where they are located mostly on GABAergic interneurons and play an important role in learning and memory. Moreover α7 nAChRs have been involved in diseases such as Alzheimer’s disease (AD) and schizophrenia, and are attracting considerable scientific interest to elucidate their contribution to disease mechanisms. While central cholinergic circuits have been investigated very extensively, the cell- and molecular biological properties of α7 nAChRs have not been studied in depth. The exact subcellular localization of α7 nAChRs is still debated, in particular in relation to synaptic sites, and only two proteins interacting with α7 nAChR, namely RIC-3 and Src-family kinases, have been identified to date. However, none of them is involved in synaptic clustering of α7 nAChR. In chapter 2 we describe the discovery of PICK1 as a novel α7 nAChR interacting protein. Thereby the PDZ domain of PICK1 binds to the large cytoplasmatic loop of α7 nAChR. We present evidence that PICK1 regulates clustering of α7 nAChRs in rat hippocampal interneurons. The more detailed investigation of α7 nAChR clustering and surface dynamics demanded the ability to express exogenous gene constructs in cultured neurons. In chapter 3 we report an optimized transfection protocol for rat hippocampal neurons. Use of the magnetofection technique allowed the parallel transfection of several constructs and their expression in neurons for up to 3 weeks in vitro. To understand the clustering and localization of α7 nAChRs it is necessary to investigate the surface dynamics of single receptors. In chapter 4 we report a detailed analysis of α7 nAChR cell surface mobility, using α-BT and QDots labeled single receptor trafficking. α7 nAChRs were found to be very mobile within the membrane. Clusters were found to be mobility traps, suggesting α7 nAChRs interact with underlying scaffolding proteins at these sites. Mobility traps were found extrasynaptically and perisynaptically in close vicinity to GABAergic and glutamatergic postsynaptic densities. While extrasynaptic α7 nAChRs might activate Ca2+-dependent signaling pathways, the perisynaptic α7 nAChRs are probably playing a modulatory role in GABAergic and glutamatergic synaptic activity. α7 nAChR mobility was not only dependent on localization but also on chronic synaptic activity changes and activation of the receptor itself. Taken together, in this thesis work we identify α7 nAChR as a highly regulated receptor. The sites of α7 nAChR-dependent Ca2+ influx are tightly controlled by the cell. α7 nAChRs are clustered at distinct sites, reflecting functional heterogeneity. We identify for the first time a direct protein-protein interaction mechanism involved in the regulation of α7 nAChR clustering and possibly surface expression. We uncover α7 nAChR clusters as sites where mobility is constrained, but single receptors are able to diffuse in and out, confirming receptor clusters as steady-state receptor aggregations. We find α7 nAChR distributed all over the cell surface with clusters formed at extra and perisynaptic sites. We speculate that α7 nAChRs have a variety of different functions dependent on their localization. The distinct mechanisms of the particular α7 nAChR subpopulations remain unclear, and are left to be addressed in future work.

Interaction of 9-amino-1,2,3,4-tetrahydroaminoacridine (THA) with human cortical nicotinic and muscarinic receptor binding in vitro

Effects of chronic sleep deprivation on central cholinergic receptors in rat brain

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.

<div>Abstract<p>The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non–small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of α5 and β3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth <i>in vitro</i> and also blocked H520 growth in nude mice <i>in vivo</i>. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC. [Cancer Res 2008;68(12):4693–700]</p></div>