Types Of Acetylcholine Receptors Research Articles

Drug resistance and neurotransmitter receptors of nematodes: recent studies on the mode of action of levamisole

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

6H,13H-Pyrazino[1,2-a;4,5-a′]diindole analogs: Probing the pharmacophore for allosteric ligands of muscarinic M2 receptors

A series of 6H,13H-pyrazino[1,2-a;4,5-a′]diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M2 receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [3H]N-methylscopolamine-occupied M2 receptors (reflected by EC50,diss) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand–receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by Ki) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M2 receptors.

In vivo characterization of a novel inhibitor of CNS nicotinic receptors

There are multiple types of nicotine acetylcholine receptors (nAChR) in the brain associated with synaptic function, signal processing, or cell survival. The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs, which may be selective for their ability to activate or inhibit a limited range of these receptor subtypes. We previously identified a family of bis-tetramethylpiperidine compounds as selective inhibitors of neuronal-type nicotinic receptors. In the present study we describe the in vivo effects and properties of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel inhibitor of neuronal nicotinic receptors. Delivered systemically, this drug can block central nervous system effects of nicotine, indicating that this drug is able to cross the blood–brain barrier and access sites in the brain. Unlike the prototype CNS-active nicotinic inhibitor, mecamylamine, TMPH blocked some but not all of the CNS effects of nicotine, indicating that it has a unique selectivity for specific receptor subtypes in the brain. The nAChR subtypes that mediate the locomotor effects and hypothermic effects of nicotine appear to be less sensitive to TMPH than those which mediate analgetic effects and discriminative stimuli. These results indicate that TMPH may possess unique selectivity for specific nicotinic receptor subtypes.

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal-type AChR with an adult-type AChR preserves myoblast fusion, muscle and end-plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end-plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.

Maternal exposure of rats to nicotine via infusion during gestation produces neurobehavioral deficits and elevated expression of glial fibrillary acidic protein in the cerebellum and CA1 subfield in the offspring at puberty

Maternal smoking during pregnancy is known to be a significant contributor to developmental neurological health problems in the offspring. In animal studies, nicotine treatment via injection during gestation has been shown to produce episodic hypoxia in the developing fetus. Nicotine delivery via mini osmotic pump, while avoiding effects due to hypoxia-ischemia, it also provides a steady level of nicotine in the plasma. In the present study timed-pregnant Sprague-Dawley rats (300–350 g) were treated with nicotine (3.3 mg/kg, in bacteriostatic water via s.c. implantation of mini osmotic pump) from gestational days (GD) 4–20. Control animals were treated with bacteriostatic water via s.c. implantation of mini osmotic pump. Offspring on postnatal day (PND) 30 and 60, were evaluated for changes in the ligand binding for various types of nicotinic acetylcholine receptors and neuropathological alterations. Neurobehavioral evaluations for sensorimotor functions, beam-walk score, beam-walk time, incline plane and grip time response were carried out on PND 60 offspring. Beam-walk time and forepaw grip time showed significant impairments in both male and female offspring. Ligand binding densities for [ 3H]epibatidine, [ 3H]cytisine and [ 3H]α-bungarotoxin did not show any significant changes in nicotinic acetylcholine receptors subtypes in the cortex at PND 30 and 60. Histopathological evaluation using cresyl violet staining showed significant decrease in surviving Purkinje neurons in the cerebellum and a decrease in surviving neurons in the CA1 subfield of hippocampus on PND 30 and 60. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum and the CA1 subfield of hippocampus on PND 30 and 60 of both male and female offspring. These results indicate that maternal exposure to nicotine produces significant neurobehavioral deficits, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum and CA1 subfield of hippocampus of the offspring on PND 30 and 60. The results show that although 60-day-old male and female rat offspring of mothers exposed to nicotine during gestation did not differ from control in body weight gain or nicotinic acetylcholine receptors ligand binding, they exhibited significant sensorimotor deficits that were consistent with the neuropathological alterations seen in the brain. These neurobehavioral and pathological deficits indicate that maternal nicotine exposure may produce long-term adverse health effects in the offspring.

Acetylcholine and Acetylcholine Receptors in Taste Receptor Cells

Neuroactive substances play important roles as transmitters and neuromodulators. Although many of these substances and/or their receptors are known to be present in taste receptor cells and nerve fibers innervating taste buds, physiological functions of these substances are not well understood (Nagai et al., 1996). Using Ca2+ imaging and immunocytochemical techniques, we have examined the physiological responses of taste receptor cells to acetylcholine (ACh), classified the types of ACh receptors, and determined the underlying signaling mechanisms in taste receptor cells. Our results suggest that ACh may be involved in cell-to-cell communication within the taste bud and in neuromodulation of taste transduction mechanisms. In Ca 2+ -imaging study, freshly isolated taste receptor cells were loaded with Ca2+-sensitive fluorescent dye Fura-2 and intracellular Ca2+ levels were measured ratiometrically. ACh induced increases in intracellular Ca 2+ levels ([Ca 2+ ]i) in taste receptor cells of mouse, rat and mudpuppy. The magnitude of the peak Ca 2+ response to ACh was concentration-dependent with half-maximum responses around 1 µM. To determine which subtypes of ACh receptors and signaling pathway were involved, we examined the effect of receptor antagonists and inhibitors for selective pathway. Atropine (0.5 µM), a muscarinic ACh receptor antagonist, blocked the ACh response, while D-tubocurarine (250 µM), a nicotinic ACh receptor antagonist, had no effect. In addition, the phospholipase C (PLC) inhibitor U73122 (5 µM) and the Ca 2+ -ATPase inhibitor thapsigargin (1 µM), which depletes intracellular Ca 2+ stores, blocked the ACh responses. These results suggest that ACh binds to muscarinic ACh receptors, which activates PLC, resulting in the production of IP3 and the subsequent release of Ca 2+ from the IP3-sensitive-intracellular stores. Since it is known that binding of ACh to muscarinic receptor subtypes M1/M3/M5 activates the PLC signaling pathways (Caulfield, 1993), our data indicates the presence of at least one of these receptors in taste receptor cells. Additionally, we found that prolonged stimulation (>1 min) with ACh (10 µM) induced a biphasic response with a transient followed by a sustained [Ca 2+ ]i increase. The sustained phase of the [Ca 2+ ]i increase was dependent on Ca 2+ influx as removal of extracellular Ca 2+ eliminated the response. Subsequently adding external Ca 2+ induced increases in [Ca2+] i , suggesting Ca2+ entry through Ca2+permeable channels. This is consistent with our previous studies showing presence of Ca 2+ store-operated channels (SOC) in taste cells (Ogura, 2002; Ogura et al., 2002). SOCs are activated solely by store depletion without requirement of a receptor-mediated mechanism, a mechanism also known as ‘capacitative calcium entry’. Thus it is possible that the sustained part of the ACh-induced calcium response is mediated by Ca 2+ influx through SOCs. In immunocytochemical study, sections containing rat circumvallate and foliate papillae were immunoreacted with an antiserum against the M1 subtype of muscarinic ACh receptors. Positive reaction was observed in many taste cells of each taste bud. In crosssections of rat circumvallate papillae, roughly half of the taste cells were immunolabeled. No selective labeling was observed in control sections, in which primary antibody was omitted. Preabsorption with antigen significantly reduced the labeling. This result suggests that taste receptor cells express M1 subtype of ACh receptor. Thus ACh can bind to the M1 subtype of the muscarinic receptors and activate the PLC/IP3 pathway. To study whether ACh is stored in synaptic vesicles in taste receptor cells and/or adjacent nerve fibers, we immunolabeled the vesicular ACh transporter (VAChT), a key element of AChcontaining vesicle in mouse taste tissue. A subset of taste receptor cells exhibited positive immunoreactivity to the antibody against VAChT. In addition, certain nerve fibers surrounding or within taste buds are positively reacted to antibodies against VAChT. These results suggest that taste receptor cells could release ACh for cell-tocell communications among taste receptor cells and/or synaptic transmission from taste receptor cells to taste sensory fibers. The presence of VAChT in adjacent nerve fibers also reveals a possibility of cholinergic modulations of taste receptor cells via the muscarinic receptors. Taken together, our results demonstrate ACh responses and its signaling pathway in taste receptor cells. Since ACh increases [Ca 2+ ]i via PLC-mediated pathway, ACh may regulate taste responses by means of changing [Ca 2+ ]i levels or PLC signaling. It is known that

Potentiation of human α4β2 neuronal nicotinic receptors by a Flustra foliacea metabolite

The effects of various Flustra foliacea metabolites on different types of human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated. Whereas most of the compounds tested had a small blocking effect, one of them, deformylflustrabromine, selectively increased the current obtained in α4β2 receptors when co-applied with acetylcholine (ACh). The current increase was reversible and concentration-dependent. This potentiating effect was still present at saturating concentrations of acetylcholine, and no changes in single-channel conductance or reversal potential were observed, thus suggesting a modification in the gating of α4β2 receptors. Dwell time analysis of single channel records indicates that the mechanism of action of deformylflustrabromine could be both an increase of the opening rate constant and a decrease of the closing rate constant on α4β2 receptors. Thus, deformylflustrabromine may constitute an excellent starting point for the future development of related agents able to potentiate human neuronal nicotinic receptor function.

Evidence for the involvement of neurotrophins in muscle transdifferentiation and acetylcholine receptor transformation in the esophagus of Myf5−/−:MyoD−/− and NT‐3−/− embryos

The primary aim of our study was to determine whether the esophageal innervation (i.e., vagal and enteric) and the skeletal muscle-secreted neurotrophins have a role in smooth-to-skeletal muscle transdifferentiation and in the muscarinic-to-nicotinic acetylcholine receptor type transition. To that end, we used genetically engineered embryos and immunohistochemistry. We found that, in the absence of Myf5 and MyoD, the esophageal muscle cells failed to develop the striated phenotype of acetylcholine receptors. In addition, the development of vagal and enteric innervation was delayed in Myf5(-/-):MyoD(-/-) and NT-3(-/-) mutants, but it was reestablished 2 days before the end of gestation. The smooth muscle cells in the esophagus appeared to be a distinct subpopulation of cells and their ability to transdifferentiate was based on their competence to express neurotrophins and their receptors. Finally, our data suggest a role for NT-3 in the esophageal muscle transdifferentiation.

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

Nicotinergic agents can act as both chemokines and chemoattractants for cell migration. Epidermal keratinocytes both synthesize acetylcholine and use it as a paracrine and autocrine regulator of cell motility. To gain a mechanistic insight into nicotinergic control of keratinocyte motility, we determined types of nicotinic acetylcholine receptors and signaling pathways regulating keratinocyte chemokinesis and chemotaxis, using respective modifications of the agarose gel keratinocyte outgrowth assay. Random migration of keratinocytes was significantly (P<0.05) inhibited by hemicholinum-3, a metabolic inhibitor of acetylcholine synthesis, as well as by the alpha-conotoxins MII and AuIB, preferentially blocking alpha3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the alpha3beta2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of alpha3beta2 included activation of the protein-kinase-C isoform delta and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of alpha7 nicotinic acetylcholine receptor. The alpha7-preferring antagonist alpha-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the alpha7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-alpha7 antisense oligonucleotides and alpha7-knockout mice. The signaling pathway mediating alpha7-dependent keratinocyte chemotaxis included intracellular calcium, activation of calcium/calmodulin-dependent protein-kinase II, conventional isoforms of protein-kinase C, phosphatidylinositol-3-kinase and engagement of Rac/Cdc42. Redistribution of alpha7 immunoreactivity to the leading edge of keratinocytes upon exposure to a chemoattractant preceded crescent shape formation and directional migration. Application of high-resolution deconvolution microscopy demonstrated that, on the cell membrane of keratinocytes, the nicotinic acetylcholine receptor subunits localize with the integrin beta1. The obtained results demonstrate for the first time that alpha3 and alpha7 nicotinic acetylcholine receptors regulate keratinocyte chemokinesis and chemotaxis, respectively, and identify signaling pathways mediating these functions, which has clinical implications for wound healing and control of cancer metastases.

Smoking is Not Beneficial for Tuberculosis

Although the recent editorial (1) accompanying the report by Blanchet and coworkers (2) states that smoking may be protective or inhibit the development of granulomatous lung disease by virtue of its anti–tumor necrosis factor(TNF) effect, it did not mention the detrimental effects of smoking on the most common granulomatous lung disease: tuberculosis. Studies from the United Kingdom, China, and, most recently, two large studies from India and Hong Kong (3, 4) have highlighted the significantly increased risk of developing active tuberculosis in smokers. Moreover, the prevalence of tuberculosis correlated with the degree and duration of tobacco use (3). The underlying mechanisms for the increased prevalence of tuberculosis in smokers have recently been elucidated. Clues about the immunosuppressive effects of tobacco smoke first came from a report showing that the parasympathetic nervous system, signaling via the vagus nerve, mediates a major, previously unnoticed, antiinflammatory pathway (5). Acetylcholinergic efferents in the vagus nerve signal to peripheral tissues via nicotinic acetylcholine receptors on macrophages, and this signal turns off production of proinflammatory cytokines such as TNF(5). Subsequently, the same group showed that acetylcholine released from the vagal efferent nerve terminals abrogates macrophagederived TNFproduction by binding to a specific type of nicotinic acetylcholine receptor ( 7), which is expressed at high levels on macrophages (6). TNFis quintessential for protective host immunity in tuberculosis, and treatment of patients with rheumatoid arthritis with monoclonal TNFinhibitors may reactivate latent tuberculosis (7). Thus, clues from the effects of smoking on inflammatory lung disorders converge on the hypothesis that smoking might oppose release or function of TNFin the lungs. Compared with other granulomatous lung diseases like sarcoidosis and hypersensitivity pneumonitis, where TNFis important in the pathogenesis, pulmonary tuberculosis causes far greater mortality (two million deaths annually) and morbidity, with consequent chronic respiratory disability due to lung remodeling (fibrosis, posttuberculous bronchiectasis, and residual cavitation). It would be more appropriate to state that the relationship of smoking to granulomatous lung disease is a doubleedged sword; the anti–TNFproperties of nicotine may be beneficial in some cases, but are clearly deleterious in others.

Bisquaternary caracurine V and iso-caracurine V salts as ligands for the muscle type of nicotinic acetylcholine receptors: SAR and QSAR studies

The binding constants (Ki values) of 24 caracurine V and 6 iso-caracurine V analogues for the muscle type of nicotinic ACh receptors (nAChR) from Torpedo californica were determined in a binding assay using (±)-[3H]epibatidine as a radioligand. The allyl alcohol group present in the iso-caracurine V ring system was found to be essential for high binding affinity. The most potent compounds are the dimethyl and di-(4-nitrobenzyl)-iso-caracurinium V salts 29 (18nM), and 31 (79nM), respectively. Compound 29 and the corresponding diallyl analogue 30 (350nM) exhibited similar binding affinities as the equally substituted neuromuscular-blocking agents toxiferine I (14nM) and alcuronium (234nM), respectively. The SAR results were confirmed by QSAR studies, which additionally revealed that the presence of hydrogen-bond acceptor groups close to the quaternary nitrogen, is detrimental for the nicotinic binding affinity. The diallyl- and dimethylcaracurinium V salts 13 and 27, respectively, which are known to be among the most potent allosteric modulators of M2 receptors (EC50=10 and 8nM, respectively), exhibited rather low nicotinic binding affinities for muscle type nAChR (Ki=1.5 and 5.2μM, respectively). Such a large difference in affinity suggests that it is possible to develop compounds with high muscarinic allosteric potency and low or negligible affinities for (α1)2β1γδ nAChR. Additionally, the iso-caracurine V analogues with binding affinities comparable to those of (+)-tubocurarine and alcuronium could become a new class of neuromuscular-blocking agents.

Acetylcholine receptors in cutaneous biology – receptorology and therapeutic implications

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.

Gating dynamics of the acetylcholine receptor extracellular domain.

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

The Intrathymic Pathogenesis of Myasthenia Gravis

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis, an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, its role is yet to be defined. The studies described herein summarize our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in the immunopathogenesis of this autoimmune disease. We review the work characterizing the expression of neuromuscular ACh receptors in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels

Acetylcholine receptor type ligand-gated ion channels are well known in animals. Homologs are identified in prokaryotes that may act as chemotactic receptors.

Molecular mechanisms of inhibition of nicotinic acetylcholine receptors by tricyclic antidepressants

In addition to their well known actions on monoamine reuptake, tricyclic antidepressants have been shown to modulate ligand-gated ion channels (LGICs). Since the muscle nicotinic acetylcholine receptor (AChR) has been the model for studying structure–function relationships of LGICs, we analyzed the action of tricyclic antidepressants on this type of AChR at both single-channel and macroscopic current levels. We also determined their effects on ACh equilibrium binding and their interactions with the different conformational states of the AChR. Antidepressants produce a significant concentration-dependent decrease in the duration of clusters of single-channels (eight fold at 20 μM). They also decrease the peak amplitude and increase the decay rate of currents elicited by rapid perfusion of ACh to outside-out patches. In equilibrium binding assays, antidepressants promote the typical high-affinity desensitized state and inhibit binding of [piperidyl-3,4- 3H ( N)]-( N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([ 3H]TCP) to its locus in resting and desensitized AChRs. The results indicate that tricyclic antidepressants: (i) interact with resting (closed), open, and desensitized channels; (ii) do not affect significantly channel opening and closing rates; (iii) do not act as fast open-channel blockers; (iv) inhibit activation of resting channels; and (v) may increase the rate of long-lived desensitization from the open state.

Frequency of Anti-AChR ϵ Subunit-specific Antibodies in MG

A definite diagnosis of myasthenia gravis (MG) relies heavily on acetylcholine receptor (AChR) antibody testing. The relatively high number of antibody-negative patients therefore, causes frequent uncertainty in confirming the diagnosis. We evaluated the sensitivity and specificity of a new, commercially available AChR antibody test that uses an approximately equal mixture of AChR from TE671-ϵ (adult type) and TE671-γ (fetal type) cells. This assay was used to re-examine 365 seronegative MG sera in which AChR antibody had not been detected by the standard assay that uses fetal type AChR. The new assay detected anti-AChR antibodies in 17 (15.5%) of 110 patients with ocular type and in 33 (12.9%) of 255 patients with generalized type MG. Anti-AChR ϵ subunit-specific antibodies were present in 13.7% of the patients in whom no AChR antibody had been detected by the standard assay, showing an increase from 79 to 82% in overall diagnostic sensitivity.

Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors.

Two functional types of nicotinic acetylcholine receptors (nAChRs) are expressed when human embryonic kidney cells are permanently transfected with equal amounts of human alpha4 and beta2 subunit cDNAs. Most (82%) of these nAChRs exhibit an EC(50) of 74 +/- 6 microM for ACh, a much lower sensitivity than the remaining fraction (EC(50) of 0.7 +/- 0.4 microM) or than expected from expression of equal amounts of alpha4 and beta2 mRNAs in Xenopus laevis oocytes. We have found three conditions that can increase the number of nAChRs with high sensitivity to activation. These are: 1) transient transfection with additional beta2 subunits, 2) overnight incubation in nicotine, or 3) overnight culture at 29 degrees C. Using metabolic labeling with [(35)S]methionine to measure subunit stoichiometry, we found that the majority of nAChRs had a stoichiometry of (alpha4)(3)(beta2)(2). Overnight treatment with nicotine increased the number of nAChRs and increased the proportion of the (alpha4)(2)(beta2)(3) stoichiometry. Alternate alpha4beta2 nAChR stoichiometries with distinct functional properties raise the possibility for an interesting mode of synaptic regulation for nicotinic signaling in the mammalian brain.

Intrathymic expression of neuromuscular acetylcholine receptors and the immunpathogenesis of myasthenia gravis.

The thymus has been considered to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, the pathogenic role of the thymus still remains a mystery. The neuromuscular type of acetylcholine receptor (AChR) was the first self-protein associated with a defined autoimmune disease that was found to be expressed by thymic stromal populations. The studies described herein represent our efforts to determine how this "promiscuous" autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizating the expression of the alpha subunit of AChR (AChRalpha) in the thymus, and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium.

Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in nonneuronal locations, but their functions remain unknown. We found that keratinocyte α7 nAChR controls homeostasis and terminal differentiation of epidermal keratinocytes required for formation of the skin barrier. The effects of functional inactivation of α7 nAChR on keratinocyte cell cycle progression, differentiation, and apoptosis were studied in cell monolayers treated with α-bungarotoxin or antisense oligonucleotides and in the skin of Acra7 homozygous mice lacking α7 nAChR channels. Elimination of the α7 signaling pathway blocked nicotine-induced influx of 45Ca2+ and also inhibited terminal differentiation of these cells at the transcriptional and/or translational level. On the other hand, inhibition of the α7 nAChR pathway favored cell cycle progression. In the epidermis of α7−/− mice, the abnormalities in keratinocyte gene expression were associated with phenotypic changes characteristic of delayed epidermal turnover. The lack of α7 was associated with up-regulated expression of the α3 containing nAChR channels that lack α5 subunit, and both homomeric α9- and heteromeric α9α10-made nAChRs. Thus, this study demonstrates that ACh signaling through α7 nAChR channels controls late stages of keratinocyte development in the epidermis by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes and that these effects are mediated, at least in part, by alterations in transmembrane Ca2+ influx.