Evidence for operation of nicotinic and muscarinic acetylcholine receptor-dependent survival pathways in human coronary artery endothelial cells

Nicotinic acetylcholine receptors (nAChRs) have recently emerged as critical players in modulation of endothelial function. In particular, studies on endothelial cells from different vascular beds have shown anti-apoptotic actions of nicotinic stimulation, but whether there is actually activation of survival signaling downstream nAChR function has not been explored. In the present work we used human coronary artery endothelial cells (HCAECs) and a pharmacological approach to examine the impact of cholinergic stimulation on survival signaling pathways. Our findings show that cholinergic receptors promote activation of three typical survival routes: the phosphatidyl-inositol-3-kinase (PI3K)/AKT axis, activated downstream muscarinic and nAChRs; the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both muscarinic acetylcholine receptor (mAChR) and nAChR. Based on their sensitivity to α-bungarotoxin, nicotinic regulation of JAK2/STAT3 and ERK1/2 occurs downstream α7-nAChRs. The present findings suggest that in HCAECs the two cholinergic receptors may act concertedly to induce an efficient survival response of coronary cells when exposed to pro-apoptotic stimuli.     

Identification of cultured cells expressing ligand-gated cationic channels.

We have identified cultured cells that express ligand-gated cation channels using a simple method which may also be applied to the screening of chemical agents for their use as agonists or antagonists. This assay is based upon the observation that many ligand-gated cation channels are permeable to lithium and agonists induce the flux of lithium into the cells which contain them. Since the accumulation of intracellular lithium can alter the cell cycle, the measurement of [3H]thymidine ([3H]thy) incorporation should reflect this occurrence. This expectation was realized using the PC12 cell line which expresses neuronal-like nicotinic acetylcholine receptor (nAChR). When cholinergic agonists are applied to PC12 cells in the presence of lithium-containing buffer and cells are subsequently pulsed with [3H]thy, the radiolabel incorporation into these cells relative to controls is reduced. If cholinergic antagonists are included or if the concentration of agonist either rapidly desensitizes receptors or is insufficient to induce channel opening, the reduction in [3H]thy incorporation is not observed. This method also provides a rapid way to screen cultured cell lines for those that express ligand-gated cation channels. This assay offers the potential to be automated for the low cost screening of drugs which act upon ligand-gated ion channels.     

The lymphocytic cholinergic system and its contribution to the regulation of immune activity

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.     

Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors

High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amount of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT(3)R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical experimental conditions.     

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.     

Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium

Acetylcholine (ACh) and its receptors play a crucial role in bladder physiology. Here, we investigated the presence of muscarinic receptor subtypes (MR) and nicotinic acetylcholine receptor (nAChR) alpha-subunits in the mouse urothelium by RT-PCR and immunohistochemistry. With RT-PCR, we detected mRNAs coding for all of the five different MR subtypes and for the nicotinic receptor subunits alpha2, alpha4, alpha5, alpha6, alpha7, alpha9 and alpha10, whereas the alpha3-subunit was not expressed. Using immunohistochemistry, we localised a panel of acetylcholine receptors in the different layers of the murine bladder urothelium, with predominant appearance in the basal plasma membrane of the basal cell layer and in the apical membrane of the umbrella cells. M2R and subunit alpha9 were observed exclusively in the umbrella cells, whereas the MR subtypes 3-5 and the nAChR subunits alpha4, alpha7 and alpha10 were also detected in the intermediate and basal cell layers. The subunit alpha5 was localised only in the basal cell layer. In conclusion, the murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since these receptors have different electrophysiological and pharmacological properties, and therefore are considered to be responsible for different cellular responses to ACh, this differential distribution is expected to confer cell type-specificity of cholinergic regulation in the bladder urothelium.     

Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.     

Computational approaches to understand alpha-conotoxin interactions at neuronal nicotinic receptors.

Recent and increasing use of computational tools in the field of nicotinic receptors has led to the publication of several models of ligand-receptor interactions. These models are all based on the crystal structure at 2.7 A resolution of a protein related to the extracellular N-terminus of nicotinic acetylcholine receptors (nAChRs), the acetylcholine binding protein. In the absence of any X-ray or NMR information on nAChRs, this new structure has provided a reliable alternative to study the nAChR structure. We are now able to build homology models of the binding domain of any nAChR subtype and fit in different ligands using docking programs. This strategy has already been performed successfully for the docking of several nAChR agonists and antagonists. This minireview focuses on the interaction of alpha-conotoxins with neuronal nicotinic receptors in light of our new understanding of the receptor structure. Computational tools are expected to reveal the molecular recognition mechanisms that govern the interaction between alpha-conotoxins and neuronal nAChRs at the molecular level. An accurate determination of their binding modes on the neuronal nAChR may allow the rational design of alpha-conotoxin-based ligands with novel nAChR selectivity.     

α7-cholinergic receptor mediates vagal induction of splenic norepinephrine

Classically, sympathetic and parasympathetic systems act in opposition to maintain the physiological homeostasis. In this article, we report that both systems work together to restrain systemic inflammation in life-threatening conditions such as sepsis. This study indicates that vagus nerve and cholinergic agonists activate the sympathetic noradrenergic splenic nerve to control systemic inflammation. Unlike adrenalectomy, splenectomy and splenic neurectomy prevent the anti-inflammatory potential of both the vagus nerve and cholinergic agonists, and abrogate their potential to induce splenic and plasma norepinephrine. Splenic nerve stimulation mimics vagal and cholinergic induction of norepinephrine and re-establishes neuromodulation in α7 nicotinic acetylcholine receptor (α7nAChR)-deficient animals. Thus, vagus nerve and cholinergic agonists inhibit systemic inflammation by activating the noradrenergic splenic nerve via the α7nAChR nicotinic receptors. α7nAChR represents a unique molecular link between the parasympathetic and sympathetic system to control inflammation.     

Pharmacological Mechanisms of Cortical Enhancement Induced by the Repetitive Pairing of Visual/Cholinergic Stimulation

Repetitive visual training paired with electrical activation of cholinergic projections to the primary visual cortex (V1) induces long-term enhancement of cortical processing in response to the visual training stimulus. To better determine the receptor subtypes mediating this effect the selective pharmacological blockade of V1 nicotinic (nAChR), M1 and M2 muscarinic (mAChR) or GABAergic A (GABA_AR) receptors was performed during the training session and visual evoked potentials (VEPs) were recorded before and after training. The training session consisted of the exposure of awake, adult rats to an orientation-specific 0.12 CPD grating paired with an electrical stimulation of the basal forebrain for a duration of 1 week for 10 minutes per day. Pharmacological agents were infused intracortically during this period. The post-training VEP amplitude was significantly increased compared to the pre-training values for the trained spatial frequency and to adjacent spatial frequencies up to 0.3 CPD, suggesting a long-term increase of V1 sensitivity. This increase was totally blocked by the nAChR antagonist as well as by an M2 mAChR subtype and GABA_AR antagonist. Moreover, administration of the M2 mAChR antagonist also significantly decreased the amplitude of the control VEPs, suggesting a suppressive effect on cortical responsiveness. However, the M1 mAChR antagonist blocked the increase of the VEP amplitude only for the high spatial frequency (0.3 CPD), suggesting that M1 role was limited to the spread of the enhancement effect to a higher spatial frequency. More generally, all the drugs used did block the VEP increase at 0.3 CPD. Further, use of each of the aforementioned receptor antagonists blocked training-induced changes in gamma and beta band oscillations. These findings demonstrate that visual training coupled with cholinergic stimulation improved perceptual sensitivity by enhancing cortical responsiveness in V1. This enhancement is mainly mediated by nAChRs, M2 mAChRs and GABA_ARs. The M1 mAChR subtype appears to be involved in spreading the enhancement of V1 cortical responsiveness to adjacent neurons.     

Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR

The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Docking of alpha-cobratoxin suggests a basal conformation of the nicotinic receptor

We investigate the interactions between the long chain alpha-cobratoxin (Cbtx) and the nicotinic acetylcholine receptor using a rigid body docking procedure. The method, (i) reproduces the binding of Cbtx to Lymnea acetylcholine-binding protein (AChBP); (ii) shows that most of the structures of AChBP obtained in the presence of antagonists are compatible with Cbtx binding; and (iii) reveals a complex between Cbtx and muscle nAChR that corresponds to the basal "resting" state conformation. The structures are made available for further understanding of the allosteric transitions of the nAChR as well as for drug design.     

Acetylcholine receptors and tau phosphorylation

Alzheimer's disease (AD) is characterized by the presence, in the brain of the patients, of two aberrant structures: intracellular neurofibrillary tangles (NFTs), containing an abnormal hyperphosphorylated form of tau protein, and extracellular senile plaques (SPs), mainly composed by fibrillar amyloid beta peptide. Another feature of AD is the neurodegeneration and dysfunction of basal forebrain cholinergic system. A possible connection among those AD characteristics could occur. Thus, the purpose of this short review is to summarize the involvement of nicotinic (nAChR) and muscarinic (mAChR) receptors on tau phosphorylation, in a direct way, or through the previous interaction of some of these receptors with amyloid beta. Several studies have demonstrated that nAChR activation results in a significantly increase of tau phosphorylation, whereas mAChR activation, may prevent tau phosphorylation.     

The role of tyrosine at the ligand-binding site of the nicotinic acetylcholine receptor

Identification of the critical residues in a receptor's ligand-binding site provides valuable structural information important for understanding the basis for ligand recognition. The design of specific ligands targeted for receptor action will depend to a great extent on detailed structural knowledge of this kind. Although the nicotinic acetylcholine receptor (nAChR) is perhaps the best characterized of all receptors, the detailed configuration of the ligand-binding site remains unknown. Structural comparisons of nicotinic agonists and antagonists have long predicted a negative subsite on the receptor to interact with the positively charged alkyl-ammonium moiety common to nearly all nicotinic agents. We have used intrinsic fluorescence spectroscopic analyses together with binding studies of selectively modified peptide fragments of the nAChR to suggest that one or two invariant tyrosine residues at positions 190 and 198 on the alpha-subunit provide the critical negative subsite required for ligand binding. Tyrosines may similarly be part of the negative subsite of muscarinic receptors and other neurotransmitter receptors that bind cationic ligands.     

The lymphocytic cholinergic system and its biological function

Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.     

Mucocutaneous cholinergic system is targeted in mustard-induced vesication

The purpose of this research is to elucidate the pharmacological mechanism mediating vesicating effects of sulfur mustard (HD) and identify an antidote to its action. HD causes blisters because epithelial cells lose their attachments. Epithelial cell adhesion is under control of the local cytotransmitter acetylcholine (ACh) working through the muscarinic and nicotinic receptor, mAChR and nAChR, classes expressed by epithelial cells. In this study, nitrogen mustard (NM)-a structural analog of HD-was used to elucidate the mechanism of vesicating effects of mustards in mucocutaneous tissues. NM caused cell detachment and cholinergic agents antagonized its effect. Radioligand binding inhibition experiments showed that NM binds to the ligand-binding site of ACh receptors (AChRs) of both classes. Ligation of AChRs on the cell membrane of keratinocytes (KC) and bronchial epithelial cells (BEC) with NM increased total esterolytic activity of serine proteinases (TEASP). Antagonists of both classes of AChRs, atropine and mecamylamine, diminished NM-induced changes, suggesting that the pathobiological effects of NM on KC and BEC result from an agonist-like degradation of ligated AChRs, predominantly of the muscarinic class. Thus, biological effects of NM on cell adhesion were antagonist-like, whereas its pharmacological effect on TEASP was agonist-like. These findings support a hypothesis that pharmacologic protection from the vesicating action of HD can be achieved by using cholinergic drugs.