The non-neuronal cholinergic system in humans: expression,function and pathophysiology

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.     

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.     

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications

Abstract.  Embryonal carcinoma and embryonic stem cells have served as models to understand basic aspects of neuronal differentiation and are promising candidates for regenerative medicine. Besides being well characterized regarding the capability of embryonal carcinoma and embryonic stem cells to be precursors of different tissues, the molecular mechanisms controlling neuronal differentiation are hardly understood. Neuropeptide and neurotransmitter receptors are expressed at early stages of differentiation prior to synaptogenesis, triggering transient changes in calcium concentration and inducing neurone-specific gene expression. In vitro neuronal differentiation of embryonal carcinoma and embryonic stem cells closely resembles early neuronal development in vivo . Murine P19 EC cells are a well-characterized model for in vitro differentiation, which upon treatment with retinoic acid differentiate into neurones. Expression and activity of various receptor proteins is regulated during their differentiation. Stimulation of kinin-B2, endothelin-B, muscarinic acetylcholine, and N-methyl-D-aspartate receptors results in transient increases of intracellular free calcium concentration [Ca²⁺]_i in P19 cells undergoing neuronal differentiation, whereas embryonal cells do not respond or show a smaller change in [Ca²⁺]_i than differentiating cells. Receptor inhibition, as studied with the example of the kinin-B2 receptor, aborts neuronal maturation of P19 cells, demonstrating the crucial importance of B2 receptors during the differentiation process. Future success in obtaining desired neuronal phenotypes from pluripotent cells in vitro may offer new therapeutic perspectives for curing genetic and acquired dysfunctions of the developing and adult nervous system.     

Tyrosine Kinase Activity Is Essential for Interleukin-1β-Stimulated Production of Interleukin-6 in U373 Human Astrocytoma Cells

Abstract: Previous studies have shown that PC12 cells depend on growth factors for their survival. When deprived of growth factors, the cells undergo a dying process termed "apoptosis" (programed cell death). We show here that muscarinic agonists inhibited the apoptotic death of growth factor-deprived PC12M1 cells (PC12 cells stably expressing cloned m1 muscarinic acetylcholine receptors). This protective effect of the muscarinic agonists was observed in both proliferating and neuronal PC12M1 cells, was blocked by the muscarinic antagonist atropine, and was not observed in PC12 cells lacking m1 receptors. Muscarinic receptors therefore mediate inhibition of apoptosis in these cells. In addition to its effect on survival, the muscarinic agonist oxotremorine induced inhibition of DNA synthesis as well as growth arrest of exponentially growing PC12M1 cells at the S and G₂/M phases of the cell cycle. Muscarinic receptors in these cells may therefore mediate inhibition of cell cycle progression.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.     

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.     

Internalization and down-regulation of muscarinic acetylcholine receptors in cerebellar granule cells of tenascin-gene deficient mice

The expression of tenascin-C on oligodendrocytes parallels the migration of granule cells in the developing cerebellum, indicating a role for tenascin-C as a guide for granule neurons to find their proper locations. In this study, cultured cerebellar granule neurons from tenascin-C-knockout mice were used to examine the role of tenascin-C in agonist-induced muscarinic acetylcholine receptor down-regulation. Exposure of granule cells from wild-type or tenascin-C-negative mice to the muscarinic acetylcholine receptor agonist carbachol (1 mM) resulted in normal sequestration of cell-surface muscarinic acetylcholine receptors as assessed by [3H]N-methylscopolamine binding; however, down-regulation of total muscarinic acetylcholine receptors, measured with [3H]quinuclidinyl benzilate, was inhibited in granule cells from tenascin-C-negative mice. Remarkably, incubation of the tenascin-C-negative cells with the microtubule stabilizer taxol (10 microM) restored down-regulation of total muscarinic acetylcholine receptors to normal levels. We speculate that agonist-induced down-regulation of muscarinic acetylcholine receptors is functionally associated with tenascin-C-regulated microtubule structures in the developing cerebellum.     

Modulation of neuritogenesis by astrocyte muscarinic receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.     

Growth Factor-Like Effects Mediated by Muscarinic Receptors in PC12M1 Cells

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.     

Specification of motoneurons from human embryonic stem cells

An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube-like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro-generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.     

Cellular Acetylcholine Content and Neuronal Differentiation

Abstract: N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma × glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.     

Development of cholinergic neurons of the rat retina

Biochemical and electrophysiological features of cholinergic neurons from the developing rat retina were analyzed in an attempt to identify important periods in the maturation of these neurons. Acetylcholine synthesis was assayed in intact, isolated retinas; choline acetyltransferase activity was measured in homogenates. Spontaneous and evoked acetylcholine release and certain aspects of synapse formation were examined in a retina-muscle cell culture system. In this system, retinal cells from rats of different ages were dissociated with trypsin and added to previously cultured rat striated muscle cells which served as postsynaptic targets for cholinergic neurons. The results indicate that two developmental periods can be described. The first stage occurs during the final week of gestation well before morphological signs of synapses appear. During this early period, cholinergic neurons acquire the ability to synthesize and release acetylcholine and to form functional synapses. These developing neurons progressively lose their ability to form and to maintain synapses with an inappropriate target. A second stage of maturation begins at the end of the first postnatal week. In this phase, dramatic increases in acetylcholine synthesis, choline acetyltransferase activity, and high-affinity choline uptake are coupled temporally with the morphological differentiation of synapses.     

Culture of chick embryo neural retina in serum-free medium : Expression of cholinergic proteins

Dissociated retinal cells from 8-day-old chick embryos were cultivated in serum-containing and in defined serum-free media. Under the latter conditions, and using polylysine as a substrate, the proliferation of glial cells was almost completely prevented, and pure (>90%) neuronal cultures could be maintained for up to 7 days in vitro. The specific but not the total activities of choline acetyltransferase and of the nicotinic and the muscarinic acetylcholine receptor were increased under serum-free culture conditions. Autoradiographic studies with [¹²⁵I]α-bungarotoxin, a selective ligand for nicotinic cholinergic receptors, showed that serum-free culture conditions may constitute a useful tool for identifying biochemically different types of retinal neurons in tissue culture.     

Muscarinic Stimulation Promotes Cultured Purkinje Cell Survival: A Role for Acetylcholine in Cerebellar Development?

Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µ M ) and pirenzepine (10 n M ), but not by methoctramine (25 n M ). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.