Acetylcholine Receptor Alpha Research Articles

New Analogues of Epiboxidine Incorporating the 4,5‐Dihydroisoxazole Nucleus: Synthesis, Binding Affinity at Neuronal Nicotinic Acetylcholine Receptors, and Molecular Modeling Investigations

A group of novel 4,5-dihydro-3-methylisoxazolyl derivatives, structurally related to epiboxidine (=(1R,4S,6S)-6-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]heptane), was prepared via 1,3-dipolar cycloaddition of acetonitrile oxide to different olefins. Target compounds 1a and 1b, 2a and 2b, 3, 4, and 5 were tested for affinity at neuronal nicotinic heteromeric (alpha4beta2) and homomeric (alpha7) acetylcholine receptors. Notably, diastereoisomers 1a and 1b were characterized by a massive drop of the affinity at the alpha4beta2 subtypes (K(i) values spanning the range 4.3-126 microM), when compared with that of epiboxidine (K(i)=0.6 nM). Therefore, the replacement of the 3-methylisoxazole ring of epiboxidine with the 4,5-dihydro-3-methylisoxazole nucleus is detrimental for the affinity at alpha4beta2 receptors. A comparable lack of affinity/selectivity for the two nAChR subtypes under study was evidenced for the remaining epiboxidine-related dihydroisoxazole derivatives 2a and 2b, and 3-5. Diastereoisomers 1a and 1b, and spirocyclic derivative 3 were docked into molecular models of the receptor subtypes under study, and their binding mode was compared with that of reference ligands endowed with high binding affinity.

Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

BackgroundDevelopment of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.Principal FindingsWe report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.ConclusionMeltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

A rapid, fluorescence‐based assay for detecting antigenic modulation of the acetylcholine receptor on human cell lines

Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation. AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells. Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG.

Association of dopamine, serotonin, and nicotinic gene polymorphisms with methylphenidate response in ADHD

Gene polymorphisms of the 3' untranslated region (3'-UTR) of the dopamine transporter (DAT1), Dopamine receptor exon 3 D4 variable number tandem repeat (DRD4VNTR), nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) and serotonin transporter promoter (SLC6A4-5HTTLPR) are under consideration as potential risk factors for attention-deficit/hyperactivity disorder (ADHD). A post-hoc attempt was made to investigate the association between the allelic variations of these candidate genes and retrospective parental report of response to methylphenidate in an ADHD-enriched, population-based twin sample. Subjects (N = 243) were selected from the twin sample based on parent report that the child had been treated with methylphenidate for ADHD symptoms. The functional polymorphisms screened were the VNTR located in the 3'-UTR of the dopamine transporter, DRD4 VNTR, CHRNA4 (rs1044396 and rs6090384) and the long (L(A) and L(G)) and short (S) forms of the serotonin transporter promoter region. Logistic regression did not demonstrate a significant association between methylphenidate treatment response and the relevant polymorphisms. The sample size had high power to detect effect sizes similar to those reported in some prior methylphenidate pharmacogenetic studies; however, the categorical (yes/no) measure of parent-reported treatment response may not have been sensitive enough to pick up statistically significant differences in treatment response based on genotype. Further studies including quantitative measures of treatment response are warranted.

The selective α7 nicotinic acetylcholine receptor agonist A-582941 activates immediate early genes in limbic regions of the forebrain: Differential effects in the juvenile and adult rat

Due to the cognitive-enhancing properties of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists, they have attracted interest for the treatment of cognitive disturbances in schizophrenia. Schizophrenia typically presents in late adolescence or early adulthood. It is therefore important to study whether alpha7 nAChR stimulation activates brain regions involved in cognition in juvenile as well as adult individuals. Here, we compared the effects of the novel and selective alpha7 nAChR agonist 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) in the juvenile and adult rat forebrain using two markers, activity-regulated cytoskeleton-associated protein (Arc) and c-Fos, to map neuronal activity. Acute administration of A-582941 (1, 3, 10 mg/kg) induced a dose-dependent increase in Arc mRNA expression in the medial prefrontal cortex (mPFC) and the ventral/lateral orbitofrontal (VO/LO) cortex of juvenile, but not adult rats. This effect was mitigated by the alpha7 nAChR antagonist methyllycaconitine. A-582941 also increased c-Fos mRNA expression in the mPFC of juvenile, but not adult rats. Furthermore, A-582941 increased the number of Arc and c-Fos immunopositive cells in the mPFC, VO/LO, and shell of the nucleus accumbens, in both juvenile and adult rats. The A-582941-induced c-Fos protein expression was significantly greater in the mPFC and VO/LO of juvenile compared with adult rats. These data indicate that A-582941-induced alpha7 nAChR stimulation activates brain regions critically involved in working memory and attention. Furthermore, this effect is more pronounced in juvenile than adult rats, indicating that the juvenile forebrain is more responsive to alpha7 nAChR stimulation. This observation may be relevant in the treatment of juvenile-onset schizophrenia.

A New Mouse Model of Autoimmune Ocular Myasthenia Gravis

To establish a novel model of autoimmune ocular myasthenia gravis (oMG) in mice and study the pathogenic mechanisms of oMG. oMG was induced in HLA-DQ8 transgenic, HLA-DR3 transgenic, major histocompatibility complex (MHC) class II-deficient, C57BL/6, and C57BL/10 mice by immunization with an Escherichia coli plasmid expressing the recombinant human acetylcholine receptor (AChR) alpha subunit. All strains of immunized mice developed ocular myasthenia gravis with varying disease incidence and severity. HLA-DQ8 transgenic mice were highly susceptible to oMG. Mice with oMG had serum autoantibodies to the mouse extraocular AChR, pathologic deposits of IgG, C3, and C5b-C9 in their extraocular and limb neuromuscular junctions, and droopiness of eyelids. HLA-DR3 transgenic and MHC class II-deficient mice were relatively resistant to oMG induced by AChR alpha subunit immunization and had minimal ocular abnormalities. These findings suggest that oMG pathogenesis could be triggered by immunity to the human AChR alpha subunit and that MHC class II molecule is required for human AChR alpha subunit presentation and CD4 cell-mediated anti-AChR antibody class switching. Differential oMG susceptibility observed in DQ8 and DR3 transgenic mice correlated with the intensity of lymphocytes to respond to the human AChR alpha subunit. This new model of oMG will be a valuable tool for studying the mechanism of oMG and gMG pathogenesis in humans and for preclinical therapeutic analysis.

Induction of antibodies to particular sites of the α7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).

Adaptive changes in structure of skeletal muscles from adult Sod1 homozygous knockout mice

Cu/Zn superoxide dismutase (SOD1), which is localized cytoplasmically and in the mitochondrial intermembrane space, is an enzyme that is critically important for superoxide free-radical elimination. Compared with age-matched wild-type littermates (Sod1 ( +/+ )), SOD1 homozygous knockout (Sod1 ( -/- )) mice have smaller body masses, heart and skeletal muscle masses, and muscle cross-sectional areas. At the light-microscopic level, cross sections of skeletal muscles from Sod1 ( -/- ) mice show no gross structural abnormalities. Following the staining of muscles of Sod1 ( -/- ) mice for succinate dehydrogenase (SDH) enzymatic activity, a grouping of SDH-positive fibers has been observed. Immunostaining for neural cell adhesion marker in the gastrocnemius muscle of Sod1 ( -/- ) mice has revealed a small number of atrophic denervated muscle fibers. No denervated fibers are observed in extensor digitorum longus (EDL), tibialis anterior, or plantaris muscles. An increase in mRNA expression levels of myogenin and acetylcholine receptor alpha has been detected in muscles in Sod1 ( -/- ) mice, but no changes in MyoD expression occur. Compared with fast oxidative fibers in EDL muscles of Sod1 ( +/+ ) mice, those of Sod1 ( -/- ) mice show increased accumulations of sub-sarcolemmal mitochondria. We conclude that the lack of SOD1 in adult Sod1 ( -/- ) mice does not result in extensive denervation of skeletal muscle fibers, although the distribution of fiber types is modified, and that fast oxidative fibers develop alterations in the amount and spatial distribution of sub-sarcolemmal mitochondria.

Detection of alpha7 acetylcholine receptor expression and activity during differentiation of pc12 cells to neurons

Detection of alpha7 acetylcholine receptor expression and activity during differentiation of pc12 cells to neurons

Autoimmune Channelopathies and Related Neurological Disorders

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

Non-Competitive Inhibitory Activities of Morphinan and Morphine Derivatives at the α3β4 Neuronal Nicotinic Acetylcholine Receptor Determined Using Nonlinear Chromatography and Chemometric Techniques

A series of morphine and morphinan derivatives were chromatographed on a column containing immobilized cellular membranes from a cell line expressing the alpha 3 beta 4 neuronal nicotinic acetylcholine receptor (alpha 3 beta 4 nAChR). The results were analyzed using chemometric and molecular modeling techniques in order to predict the noncompetitive inhibitory (NCI) activity of these compounds, the molecular basis for the predicted activity and the binding sites of the inhibitors. The data demonstrated that seven of seven morphinans were NCIs and bound in the central lumen of the nAChR while only 2 of 13 morphine derivatives had NCI activity and these compounds most likely bound at the quinacrine binding site on the nAChR. The predicted activities were confirmed using functional inhibition studies. The results indicate that this approach can be used to rapidly assess pharmacological activity and to guide new drug design.

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.

Slow-channel mutation in acetylcholine receptor alphaM4 domain and its efficient knockdown.

To identify the genetic basis of a slow-channel myasthenic syndrome, characterize functional properties of the mutant receptor, and selectively silence the mutant allele. We performed nutation analysis, cloning, and patch-clamp analysis of the functional properties of the mutant receptor; screening for a small interfering RNA with check plasmid; and assessed of the efficacy of small interfering RNA at the messenger RNA, protein, and functional levels. We traced the cause of a slow-channel myasthenic syndrome to a C418W mutation in the M4 domain of the acetylcholine receptor alpha subunit. The mutation is the first one to occur spontaneously in an M4 domain of the receptor, and it is positioned within a stripe of hydrophobic residues facing the lipid bilayer. Kinetic analysis shows that alphaC418W enhances the channel opening equilibrium constant 26-fold without altering agonist affinity. Using a check plasmid as a screening tool, we identified a small interfering RNA that markedly suppresses the mutant but not the wild-type allele at the messenger RNA, protein, and functional levels. alphaC418W occurring in humans causes a slow-channel syndrome by enhancing the relative stability of the channel open state. Efficient and selective knockdown of the mutant allele holds promise of therapeutic gene silencing.

Mechanism of Cl- Selection by a Glutamate-gated Chloride (GluCl) Receptor Revealed through Mutations in the Selectivity Filter

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClbeta receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClbeta receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P(Cl)/P(Na) ratios, but the GluClbeta receptor's mutants retained high capacity to select Cl(-) over Na(+). These results indicate that, unlike the proposed case of anionic Gly- and gamma-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClbeta receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClbeta receptor's channel, Cl(-) interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.

Potentiation of Acetylcholine Receptors by Divalent Cations: Fig. 1.

Divalent cations promote activation of several nicotinic acetylcholine receptor (AChR) subtypes, presumably by lowering the energetic barrier between open and closed conformations. In wild-type alpha7 AChRs, binding of calcium to a particular part of the extracellular domain is required for potentiating activation. McLaughlin et al. (p. 16) tested the hypothesis that movements involved in agonist activation and calcium modulation involve a nearby beta sheet by linking strands within this sheet through disulfide bonds formed by replacing adjacent amino acids with cysteines to alter its mobility. These studies are helping to reveal how movements initiated by agonist binding to ACh binding sites are propagated through the extracellular domain of AChRs to regulate opening of the cation channel through the membrane.

Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

alpha-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick alpha7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu172) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby beta-sheet formed by the beta7, beta9, and beta10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the beta7 and beta10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the beta7/beta9/beta10-sheet as a result of introduction of a disulfide bond between the beta strands would alter activation by agonists. Using a nondesensitizing alpha7 mutant background (L247T), we identified one mutant pair, K144C + T198C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca2+, Ba2+, or Sr2+) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the alpha7 antagonist methyllycaconitine and were abolished when Glu172 was mutated to glutamine. When the K144C + T198C pair was expressed in wild-type alpha7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into beta7/beta9/beta10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the beta-sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.

A steroid hormone affects sodium channel expression in Manduca central neurons

Neuronal differentiation is characterized by stereotypical sequences of membrane channel and receptor acquisition. This is regulated by the coordinated interactions of a variety of developmental mechanisms, one of which is the control by steroid hormones. We have used the metamorphosis of the holometabolous insect, Manduca sexta, as a model to study effects of 20-hydroxyecdysone on the maturation of thoracic neuron membrane channel expression. To test for direct hormone action, neurons were dissociated into primary cell culture on the first day of pupal life. In situ hybridization demonstrated that the amount of expression of the acetylcholine receptor alpha subunit, MARA1, was not affected by 20-hydroxyecdysone. Immunocytochemistry with an antibody directed against the SP19 segment of voltage-gated sodium channels revealed no effect of 20-hydroxyecdysone treatment during the first 6 days in culture. SP19 sodium channel protein was evenly distributed along all neurites. In contrast, after 8 days in culture, 20-hydroxyecdysone increased the amount of SP19 protein expression and strongly affected its distribution in differentiating neurons. In the presence of 20-hydroxyecdysone, patches of high densities of SP19 sodium channel protein were found in growth cones close to the base of filopodia. This is a further step toward unraveling the blend of membrane proteins under the control of steroids during the development of the central nervous system of postembryonic Manduca. Our results, taken together with previous studies, indicate that 20-hydroxyecdysone does not affect the expression of potassium membrane current or of the nicotinic acetylcholine receptor but instead regulates the amplitude of the calcium membrane current and the amount and distribution of SP19 sodium channel protein.

Succinylcholine-induced Hyperkalemia in Acquired Pathologic States

Lethal hyperkalemic response to succinylcholine continues to be reported, but the molecular mechanisms for the hyperkalemia have not been completely elucidated. In the normal innervated mature muscle, the acetylcholine receptors (AChRs) are located only in the junctional area. In certain pathologic states, including upper or lower motor denervation, chemical denervation by muscle relaxants, drugs, or toxins, immobilization, infection, direct muscle trauma, muscle tumor, or muscle inflammation, and/or burn injury, there is up-regulation (increase) of AChRs spreading throughout the muscle membrane, with the additional expression of two new isoforms of AChRs. The depolarization of these AChRs that are spread throughout the muscle membrane by succinylcholine and its metabolites leads to potassium efflux from the muscle, leading to hyperkalemia. The nicotinic (neuronal) alpha7 acetylcholine receptors, recently described to be expressed in muscle also, can be depolarized not only by acetylcholine and succinylcholine but also by choline, persistently, and possibly play a critical role in the hyperkalemic response to succinylcholine in patients with up-regulated AChRs.

Immunologic Role of the Cholinergic Anti‐Inflammatory Pathway and the Nicotinic Acetylcholine α7 Receptor

Cytokines are small protein molecules that facilitate communication between cells of the immune system and other tissues. Their messenger function elicits responses in other cells, ranging from DNA binding and tissue remodeling to coordinating the local cellular response to inflammation. The magnitude of the cytokine response is regulated closely, because an over- or underabundance of cytokine activity can impair organ function and cause shock and tissue injury. Counterregulatory molecular and humoral mechanisms protect the host from cytokine excess, including the pituitary-adrenal-glucocorticoid system, and the anti-inflammatory cytokine system. These humoral systems are protective, but they are relatively slow-acting, concentration-gradient dependent, and not integrated. We recently discovered that the "cholinergic anti-inflammatory pathway," a nervous system-based, rapid, and locally acting mechanism, can inhibit the cytokine response. Signals transmitted via the vagus nerve converge on cytokine-producing cells that express the nicotinic acetylcholine receptor alpha 7 (nAChR alpha7). The alpha7 receptor is an essential component of the cholinergic anti-inflammatory pathway, because activation of this receptor prevents cytokine release. Advances in understanding the molecular structure and function of alpha7 have begun to provide a better understanding of potential mechanisms regarding its assembly, expression, and cytokine-inhibiting functions. It may be possible to exploit this pathway to therapeutic advantage for diseases caused by excessive cytokine activity.