Storage Of Acetylcholine Research Articles

WS3.7. Repetitive Nerve Stimulation Testing

Repetitive nerve stimulation (RNS) is the most frequently used electrodiagnostic test to diagnose neuromuscular junction disorders. Although seemingly simple, attention to technique is essential to avoid technical errors. Stimulation at low rates (2–5 Hz) (LF-RNS) depletes acetylcholine stores at diseased motor endplates, causing failure of neuromuscular transmission. A decrement of at least 10% to trains of 2-3 Hz stimulation at rest or immediately after a period of maximal muscle contraction (“activation”) is the usual criterion of abnormality; 5% can be used if technical artifacts can be excluded. Take care to deliver adequate stimulation and prevent movement of the tested muscle throughout nerve stimulation; peripheral muscles should be kept warm during testing. RNS is reportedly abnormal in up to 90% of generalized myasthenia gravis (MG) patients and in up to 60% of ocular myasthenia – sensitivity is strongly dependent on the muscles selected for testing. RNS is more often abnormal in proximal, facial and clinically weak muscles; even so, RNS is frequently normal in mild or purely ocular disease. For maximal diagnostic yield, test several muscles, including those that are weak. The characteristic findings in Lambert-Eaton myasthenia (LEM) are: small CMAPs at rest, a decrement to LF-RNS, and marked facilitation. In MG, activation for 30–60 seconds is used to unmask a mild decrement. In LEM, activation is used to demonstrate facilitation; the simplest procedure is to elicit a single CMAP from the rested muscle, have the patient contract the muscle for 10 seconds, then immediately elicit a single CMAP. Facilitation of at least 60% is evidence of a pre-synaptic disorder, but may not be seen in all muscles. A decrement in at least one hand muscle is the most frequent RNS abnormality in LEM and may be seen even when CMAPs are not small and diagnostic facilitation cannot be demonstrated. In MG the decrement is usually greatest in the 4th or 5th response and is less during subsequent responses, producing a so-called ‘‘U-shaped” pattern. A continually progressive decrement is usually seen in LEM. The pattern in MuSK MG resembles that in LEM.

Physiology of cholinergic and adrenergic synapses. Role in pharmacology and practical significance

The transmission of signals between cholinergic neurons and from neurons to muscle cells (neuro-neuronal and neuromuscular transduction) occurs through synapses. They are formed by the membranes of two contacting cells, presynaptic and postsynaptic, which are separated by a narrow synaptic gap. The review article provides up-to-date information about the physiological processes in cholinergic and adrenergic synapses. The role of these synapses in pharmacology and their practical significance are presented. The transmission of excitation in cholinergic synapses occurs with the help of acetylcholine. The stages of synthesis, storage and release of acetylcholine are the same in all cholinergic neurons. The specific effects of acetylcholine mediated through cholinergic synapses depend mainly on the type of synaptic cholinergic receptors. In the system of efferent innervation, adrenergic synapses are formed by the endings of postganglionic sympathetic (adrenergic) fibers and cells of effector organs.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4× 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

Acetylcholine stored in dendritic cells plays a role in the differentiation of naïve CD4+ T cells into a Th1 phenotype

AbstractObjectiveImmune cells, particularly dendritic cells (DC), express choline acetyltransferase, an enzyme essential for the production of acetylcholine (ACh); thus, DC appear to store ACh. However, the immunological role of intracytoplasmic ACh within DC is largely unknown.MethodsTo examine the role of ACh during adaptive responses, naïve CD4+ T cells were stimulated with anti‐CD3 and anti‐CD28 antibodies in the presence or absence of ACh. Next, monocyte‐derived DC (Mo‐DC) were stained with an anti‐ACh antibody to investigate the intracytoplasmic storage of ACh.ResultsThe results showed that Mo‐DC stored ACh in the intracytoplasmic space. Stimulation with lipopolysaccharide (a Th1‐inducing agent) increased the levels of ACh in Mo‐DC; however, levels decreased under conditions that mimicked the interaction between DC and naïve CD4+ T cells. Interferon‐γ secretion increased preferentially in the presence of ACh, suggesting that naïve CD4+ T cells differentiate into T helper (Th)1 cells in the presence of ACh.ConclusionsThese observations suggest that Mo‐DC increase ACh storage in the presence of a Th1 adjuvant and release it during the interaction between DC and naïve CD4+ T cells, thereby driving their differentiation into Th1 cells. Thus, DC might utilize ACh to regulate the Th1 population in response to certain antigens.

Differential impact of genetically modulated choline transporter expression on the release of endogenous versus newly synthesized acetylcholine

The efficient import of choline into cholinergic nerve terminals by the presynaptic, high-affinity choline transporter (CHT, SLC5A7) dictates the capacity for acetylcholine (ACh) synthesis and release. Tissue levels of ACh are significantly reduced in mice heterozygous for a loss of function mutation in Slc5a7 (HET, CHT+/-), but significantly elevated in overexpressing, Slc5a7 BAC-transgenic mice (BAC). Since the readily-releasable pool of ACh is thought to constitute a small fraction of the total ACh pool, these genotype-dependent changes raised the question as to whether CHT expression or activity might preferentially influence the size of reserve pool ACh vesicles. In the current study, we approached this question by evaluating CHT genotype effects on the release of ACh from suprafused mouse forebrain slices. We treated slices from HET, BAC or wildtype (WT) controls with elevated K+ and monitored release of both newly synthesized and storage pools of ACh. Newly synthesized ACh produced following uptake of [3H]choline was quantified by scintillation spectrometry whereas release of endogenous ACh storage pools was quantified by an HPLC-MS approach, from the same samples. Whereas endogenous ACh release scaled with CHT gene dosage, preloaded [3H]ACh release displayed no significant genotype dependence. Our findings suggest that CHT protein levels preferentially impact the capacity for ACh release afforded by mobilization of reserve pool vesicles.

CXCL12 Regulates the Cholinergic Locus and CHT1 Through Akt Signaling Pathway

Background: CXCL12 is pivotal for cholinergic neurons, and it induces the expressions of several genes that are essential for synthesis and storage of acetylcholine(ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. The present study explored the impact of pharmacological Akt inhibition upon cholinergic gene expression. Methods: Western blotting was employed to determine the level of p-AKT, RT-PCR to check the mRNA levels of and CHT1(choline transporter1),VAChT and ChAT, ELISA to decipher the secretion of ACh and the activity of choline acetyltransferase. Results: Here we demonstrated, in the rat pheochromocytoma cell line PC12 and in primary rat neuronal cultures, that CXCL12-evoked up-regulation of CHT1, VAChT and ChAT was mediated by Akt. Inhibition of Akt by the pharmacological inhibitor GSK690693 eliminated CXCL12-stimulated increases in cholinergic gene expression. Moreover, treatment with GSK690693 reversed CXCL12-evoked increases in choline acetyltransferase activity and ACh production. Conclusion: Our results suggest that CXCL12 contributes to cholinergic gene expression via Akt signaling pathway.

Pulmonary inflammation is regulated by the levels of the vesicular acetylcholine transporter.

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.

Transgenic overexpression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance

The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC–CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [3H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC–CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC–CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC–CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC–CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC–CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.

Non-neuronal cholinergic machinery present in cardiomyocytes offsets hypertrophic signals

Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counteract or partially neutralize hypertrophic adrenergic effects.

New Considerations on the Neuromodulatory Role of Thiamine

Background: A nonmetabolic role for thiamine in cholinergic neurotransmission has long been suggested. The mechanism remains unclear. We sought to extend our previous research to elucidate the effect of the thiamine metabolic antagonist, oxythiamine, on the release of acetylcholine from the brain. Methods: The potassium-stimulated release of acetylcholine from superfused rat brain slices was determined. Hand-cut slices of cerebral cortex were preincubated with tritiated choline to label acetylcholine stores. Two periods of stimulation (S1, S2) with 50 mmol/l solution for 3.5 min were performed as superfusate was collected. During S1, only 50 mmol/l potassium-containing Krebs-bicarbonate buffer with 2 mmol/l calcium was used. Using a two-by-two design, S2 consisted of exposure to 50 mmol/l potassium with or without 10^–4 mol/l oxythiamine, with or without calcium. The S2/S1 ratio was calculated. Results: Oxythiamine enhanced the potassium-evoked release of acetylcholine by 60% but only when calcium was present in the superfusing medium. Conclusion: These data confirm earlier findings with oxythiamine on the calcium-mediated synaptic transmission of acetylcholine and support a possible neuromodulatory role for thiamine distinct from its actions as a cofactor during metabolic processes.

Search for the acetylcholine and vesamicol binding sites in vesicular acetylcholine transporter: the region around the lumenal end of the transport channel

Vesicular acetylcholine transporter (VAChT; TC 2.A.1.2.13) mediates storage of acetylcholine (ACh) by synaptic vesicles. A three-dimensional homology model of VAChT is available, but the binding sites for ACh and the allosteric inhibitor (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) are unknown. In previous work, mutations of invariant W331 in the lumenal beginning of transmembrane helix VIII (TM VIII) of rat VAChT led to as much as ninefold loss in equilibrium affinity for ACh and no loss in affinity for vesamicol. The current work investigates the effects of additional mutations in and around W331 and the nearby lumenal end of the substrate transport channel. Mutants of human VAChT were expressed in the PC12(A123.7) cell line and characterized using radiolabeled ligands and filtration assays for binding and transport. Properties of a new and a repeat mutation in W331 are consistent with the original observations. Of 16 additional mutations in 13 other residues (Y60 in the beginning of lumenal Loop I/II, F231 in the lumenal end of TM V, W315, M316, K317, in the lumenal end of TM VII, M320, A321, W325, A330 in lumenal Loop VII/VIII, A334 in the lumenal beginning of TM VIII, and C388, C391, F392 in the lumenal beginning of TM X), only A334F impairs binding. This mutation decreases ACh and vesamicol equilibrium binding affinities by 14- and 4-fold, respectively. The current results, combined with previous results, demonstrate existence of a spatial cluster of residues close to vesicular lumen that decreases affinity for ACh and/or vesamicol when the cluster is mutated. The cluster is composed of invariant W331, highly conserved A334, and invariant F335 in TM VIII and invariant C391 in TM X. Different models for the locations of the ACh and vesamicol binding sites relative to this cluster are discussed.

Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V max of choline uptake, but had no effect on K m for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.

Ciliary neurotrophic factor enhances nicotinic synaptic transmission in sympathetic neurons

Nicotinic acetylcholine receptors mediate fast synaptic transmission in both central and peripheral nervous systems. These receptors play important roles in various physiological functions and are involved in different neurological diseases. A disruption in nicotinic receptor-mediated synaptic transmission due to the loss of nAChRs was detected in the brains of patients with Parkinson's disease and Alzheimer's disease. Although ciliary neurotrophic factor (CNTF) has been reported to promote the cholinergic properties by increasing the production and storage of acetylcholine, it is still unclear whether CNTF can enhance nicotinic synaptic neurotransmission. In this study, we found that CNTF dramatically enhanced the frequency and amplitude of nicotinic excitatory post-synaptic currents in rat superior cervical ganglion neurons maintained in a medium supplemented with nerve growth factor. Moreover, the number of neurons displaying nicotinic synaptic currents was also significantly increased by CNTF. These results suggest that CNTF could enhance nicotinic synaptic transmission via both presynaptic and postsynaptic mechanisms. The findings of this study reinforce the rationale for the usage of combinations of different neurotrophic factors for the therapy of neurodegenerative diseases.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

Some Currently Neglected Aspects of Cholinergic Function

At the request of the organizers of the XIII International Symposium on Cholinergic Mechanisms, I have selected for review three topics not well represented at the meeting: the synthesis, storage, and release of acetylcholine at cholinergic nerve endings; cholinergic-specific antigens; and neuropeptides as cholinergic co-transmitters. The first topic is illustrated by work with two model systems, the electromotor nerve terminals of the electric ray and the myenteric plexus of the guinea-pig ileum, the second by work with Chol-1, a group of cholinergic-specific gangliosides sialylated on their N-acetylgalactosamine residues, and the third by vasoactive intestinal polypeptide which is a co-transmitter at 70% of cholinergic nerve endings in brain and is also present in both of the model cholinergic systems.

Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice

Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.

Purinergic cotransmission

Adenosine 5'-triphosphate (ATP) is a cotransmitter with classical transmitters in most nerves in the peripheral and central nervous systems, although the proportions vary between tissues and species and in different developmental and pathophysiological circumstances. There was early evidence that ATP was released together with acetylcholine (ACh) from motor nerves supplying skeletal muscle, although it was considered at the time as a molecule involved in the vesicular uptake and storage of ACh. Later it was shown that in the developing neuromuscular junction, released ATP acted on P2X receptor ion channels as a genuine cotransmitter with ACh. Adenosine triphosphate was shown to be released from sympathetic nerves supplying the guinea-pig taenia coli in 1971. Soon after, the possibility was raised that ATP was coreleased with noradrenaline from sympathetic nerves to guinea-pig seminal vesicle, cat nictitating membrane and guinea-pig vas deferens. Sympathetic purinergic cotransmission has also been demonstrated in many blood vessels. Parasympathetic nerves supplying the urinary bladder use ACh and ATP as cotransmitters; ATP acts through P2X ionotropic receptors, whereas the slower component of the response is mediated by the metabotropic muscarinic receptor. Adenosine triphosphate and glutamate appear to be cotransmitters in primary afferent sensory neurons. Adenosine triphosphate, calcitonin gene-related peptide and substance P coexist in some sensory-motor nerves. A subpopulation of intramural enteric nerves provides non-adrenergic, non-cholinergic inhibitory innervation of gut smooth muscle. Three cotransmitters are involved, namely ATP, nitric oxide and vasoactive intestinal polypeptide. In recent years, studies have shown that ATP is released with ACh, noradrenaline, glutamate, gamma-aminobutyric acid, 5-hyroxytryptamine and dopamine in different subpopulations of neurons in the central nervous system.

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway.

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.

Chagas' disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells

A parasite-derived neurotrophic factor (PDNF) produced by the Chagas' disease parasite Trypanosoma cruzi binds nerve growth factor (NGF) receptor TrkA, increasing receptor autophosphorylation, and activating phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK/Erk) pathways, and transcription factor CREB. The end-result is enhanced survival and neuritogenesis of various types of neurons. PDNF also enhances the expression and activity of tyrosine hydroxylase, a rate limiting enzyme in the synthesis of dopamine and other catecholamine neurotransmitters. It remains unknown, however, if PDNF alters expression and metabolism of acetylcholine (ACh), a neurotransmitter thought to play a role in Chagas' disease progression. Here we demonstrate that PDNF stimulates mRNA and protein expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are critical for synthesis and storage of ACh. Stimulation requires functional TrkA because it did not occur in cell mutants that lack the receptor and in TrkA-expressing wild-type cells treated with K252a, an inhibitor of TrkA kinase activity. It also requires TrkA-dependent PI3K and MAPK/Erk signaling pathways because PDNF stimulation of cholinergic transcripts is abolished by specific pharmacological inhibitors. Furthermore, the cholinergic actions of PDNF were reproduced by PDNF-expressing extracellular T. cruzi trypomastigotes at the start of host cell invasion. In contrast, host cells bearing intracellular T. cruzi showed decreased, rather than increased, cholinergic gene expression. These results suggest that T. cruzi invasion of the nervous system alters cholinergic gene expression and that could play a role in neuropathology, and/or lack thereof, in Chagas' disease patients.

Effects of muscarinic agonists in the guinea‐pig prostate

1. The contractile response to transmural stimulation of the guinea-pig prostate is largely due to activation of noradrenergic neurons but there is a small contribution from cholinergic neurons. Carbachol and acetylcholine have been reported to act via muscarinic M(1) cholinoceptors to facilitate contractions produced by neuronal stimulation of the tissue. This action of cholinomimetics was further investigated in isolated ventral lobes of the prostate. 2. Oxotremorine-M, bethanecol, pilocarpine, xanomeline and McN-A-343 produced facilitation of the response to transmural stimulation of the prostate. When carbachol was applied as the first agonist, the facilitatory response to the latter four agonists above was absent or reduced, compared with the effect observed when the other agonist was applied first, indicating that the effect of these agonists is readily desensitized. Only oxotremorine-M was unaffected by prior exposure of the tissue to carbachol. When applied first, pilocarpine and xanomeline produced a smaller degree of facilitation than carbachol indicating they were partial agonists for the effect. The facilitation produced by McN-A-343, when applied as the first agonist, was variable. In some preparations the facilitation was less than that of carbachol but in others it exceeded that produced by a subsequent application of carbachol. 3. The release of endogenous choline from the prostate was measured at rest and during transmural stimulation using a chemiluminescent technique. A statistically significant negative correlation existed between pmol mg(-1) of endogenous choline released during transmural stimulation and the mass of the ventral lobe of the prostate. As the guinea-pig prostate is known to undergo postpubertal stromal hypertrophy the finding suggests that endogenous choline release from the prostate is largely from epithelial, rather than stromal tissue. 4. The possible involvement of facilitatory M(1) autoreceptors on cholinergic neurons in the effect of cholinomimetics was investigated. Tissue was incubated with (3)H-choline to label neuronal stores of acetylcholine and the subsequent release of (3)H-choline from the tissue was measured. Carbachol per se increased the release of (3)H-choline. 5. Transmural stimulation usually increased the release of (3)H-choline but in c. 30% of preparations there was a decrease. In the presence of carbachol there was a significant increase in the release of (3)H-choline during transmural stimulation of prostate lobes. However, there was no correlation between the two effects of carbachol; facilitating contractions produced by transmural stimulation and enhancing (3)H-choline release during transmural stimulation. The finding provides no evidence that facilitatory M(1) autoreceptors on cholinergic neurons play a major role in the facilitation of contractions.