Acetylcholine Synthesis Research Articles

ACETYLCHOLINE AND ITS SYNTHESIZING ENZYME CHOLINE ACETYLTRANSPHERASE IN THE ENTERIC NERVOUS SYSTEM

The enteric nervous system is the largest and most complex division of the peripheral nervous system. Located in the wall of the gastrointestinal tract, it is a regulatory and coordination unit of the nervous system. Neurochemical, pharmacological, and functional studies describe three main classes of neurons in the enteric nervous system– primary afferent, interneurons and motor neurons. These are grouped in ganglia, which are connected and form plexuses. Acetylcholine is a major neurotransmitter that plays a pivotal role in the enteric nervous system and several non-neuronal structures. Internal cholinergic neurons and vagus terminals in the enteric nervous system use acetylcholine as the main excitatory neurotransmitter, regulating motility and mucosal function in the digestive system. The enzyme choline acetyltransferase (ChAT) that catalyzes the synthesis of acetylcholine represents the most specific cholinergic marker. Recent markers used to visualize cholinergic structures are the splicing variants of ChAT mRNA that are transcribed from the ChAT gene. Different alternatively spliced ChAT mRNA variants are transcribed in many animal species, including humans. In the mouse, seven variants in the 5’-noncoding region and two variants that differ in their coding region are described. Morphological, genetic and molecular analysis of ChAT and its splicing variants, as the most reliable and frequently used marker for cholinergic structures, would contribute to a better understanding of the physiological and pathological states of cholinergic neurons in the enteric nervous system.

In vitro and in silico inhibition performance of choline against CYP1A2, CYP2D6 and CYP3A4

Choline is an essential nutrient involved in the synthesis of acetylcholine in the cholinergic neurons. The pharmacokinetic properties of choline are well described; however, there is a lack of data about its activity toward the CYP450 superfamily of enzymes. Therefore, the aim of this study was to conduct in silico and in vitro activity assessments of choline against three major CYP450 isoforms—CYP1A2, CYP2D6, and CYP3A4. Preliminary in silico modeling was performed on a specialized DL-CYP Prediction Server to evaluate the affinity of choline toward the enzymes. The in vitro study contained specific cytochrome P450 isoform inhibitors and substrates (for CYP1A2, CYP2D6, and CYP3A4) to determine the inhibition performance of choline at five different concentrations (0.150 − 1 µmol/L). The potential interactions of choline and CYPs were displayed after molecular docking with Glide (Schrödinger). In addition, induced-fit simulations and binding free energy calculations MM/GBSA (Molecular Mechanics-Generalized Born Surface Area) were applied to predict the accessibility in each CYP isoform. The initial in silico simulations revealed that choline lacks inhibition potency against the aforementioned enzymes. The in vitro evaluations confirmed that choline possessed no effect against CYP1A2; however, at 1 µmol/L choline exerted 22% and 27% blocking capacity against CYP2D6 and CYP3A4, respectively. Furthermore, there was good correlation between the in vitro results and the complexes’ free binding energy recalculations. Overall, the assessments indicated that choline is a weak CYP2D6 and CYP3A4 inhibitor. The latter results should be considered as a source of future unwanted drug–drug interactions.

Serum unsaturated phosphatidylcholines predict longitudinal basal forebrain degeneration in Alzheimer's disease.

Basal forebrain cholinergic neurons are among the first cell types affected by Alzheimer's disease pathology, but the cause of their early vulnerability is unknown. The lipid phosphatidylcholine is an essential component of the cell membrane, and phosphatidylcholine levels have been shown to be abnormal in the blood and brain of Alzheimer's disease patients. We hypothesized that disease-related changes in phosphatidylcholine metabolism may disproportionately affect basal forebrain cholinergic neurons due to their extremely large size, plasticity in adulthood and unique reliance on phosphatidylcholine for acetylcholine synthesis. To test this hypothesis, we examined whether serum phosphatidylcholine levels predicted longitudinal basal forebrain degeneration in Alzheimer's disease. All data were collected by the Alzheimer's Disease Neuroimaging Initiative. Participants were divided into a normal CSF group (controls; n = 77) and an abnormal CSF group (preclinical and clinical Alzheimer's disease; n = 236) based on their CSF ratios of phosphorylated tau and amyloid beta at baseline. Groups were age-matched (t = 0.89, P > 0.1). Serum lipidomics data collected at baseline were clustered by chemical similarity, and enrichment analyses were used to determine whether serum levels of any lipid clusters differed between the normal and abnormal CSF groups. In a subset of patients with longitudinal structural MRI (normal CSF n = 62, abnormal CSF n = 161), two timepoints of MRI data were used to calculate grey matter annual percent change for each participant. Multivariate partial least squares analyses tested for relationships between neuroimaging and lipidomics data which are moderated by CSF pathology. Our clustering analyses produced 23 serum lipid clusters. Of these clusters, six were altered in the abnormal CSF group, including a cluster of unsaturated phosphatidylcholines. In the subset of participants with longitudinal structural MRI data, a priori nucleus basalis of Meynert partial least squares analyses detected a relationship between unsaturated phosphatidylcholines and degeneration in the nucleus basalis which is moderated by Alzheimer's disease CSF pathology (P = 0.0008). Whole-brain grey matter partial least squares analyses of all 23 lipid clusters revealed that only unsaturated phosphatidylcholines and unsaturated acylcarnitines exhibited an Alzheimer's disease-dependent relationship with longitudinal degeneration (P = 0.0022 and P = 0.0018, respectively). Only the unsaturated phosphatidylcholines predicted basal forebrain degeneration in the whole-brain analyses. Overall, this study provides in vivo evidence for a selective relationship between phosphatidylcholine and basal forebrain degeneration in human Alzheimer's disease, highlighting the importance of phosphatidylcholine to basal forebrain grey matter integrity.

Role of Vitamins in Neurodegenerative Diseases: A Review.

Vitamins are the micronutrients required for boosting the immune system and managing any future infection. Vitamins are involved in neurogenesis, a defense mechanism working in neurons, metabolic reactions, neuronal survival, and neuronal transmission. Their deficiency leads to abnormal functions in the brain like oxidative stress, mitochondrial dysfunction, accumulation of proteins (synuclein, Aβ plaques), neurodegeneration, and excitotoxicity. In this review, we have compiled various reports collected from PubMed, Scholar Google, Research gate, and Science direct. The findings were evaluated, compiled, and represented in this manuscript. The deficiency of vitamins in the body causes various neurological disorders like Alzheimer's disease, Parkinson's disease, Huntington's disease, and depression. We have discussed the role of vitamins in neurological disorders and the normal human body. Depression is linked to a deficiency of vitamin-C and vitamin B. In the case of Alzheimer's disease, there is a lack of vitamin- B1, B12, and vitamin-A, which results in Aβ-plaques. Similarly, in Parkinson's disease, vitamin- D deficiency leads to a decrease in the level of dopamine, and imbalance in vitamin D leads to accumulation of synuclein. In MS, vitamin-C and vitamin-D deficiency causes demyelination of neurons. In Huntington's disease, vitamin- C deficiency decreases the antioxidant level, enhances oxidative stress, and disrupts the glucose cycle. vitamin B5 deficiency in Huntington's disease disrupts the synthesis of acetylcholine and hormones in the brain.

Mesenchymal stromal cells alleviate acute respiratory distress syndrome through the cholinergic anti-inflammatory pathway

Mesenchymal stromal cells (MSCs) have been considered a promising alternative for treatment of acute respiratory distress syndrome (ARDS). However, there is significant heterogeneity in their therapeutic efficacy, largely owing to the incomplete understanding of the mechanisms underlying the therapeutic activities of MSCs. Here, we hypothesize that the cholinergic anti-inflammatory pathway (CAP), which is recognized as a neuroimmunological pathway, may be involved in the therapeutic mechanisms by which MSCs mitigate ARDS. Using lipopolysaccharide (LPS) and bacterial lung inflammation models, we found that inflammatory cell infiltration and Evans blue leakage were reduced and that the expression levels of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in lung tissue were significantly increased 6 hours after MSC infusion. When the vagus nerve was blocked or α7 nicotinic acetylcholine (ACh) receptor (α7nAChR)-knockout mice were used, the therapeutic effects of MSCs were significantly reduced, suggesting that the CAP may play an important role in the effects of MSCs in ARDS treatment. Our results further showed that MSC-derived prostaglandin E2 (PGE2) likely promoted ACh synthesis and release. Additionally, based on the efficacy of nAChR and α7nAChR agonists, we found that lobeline, the nicotinic cholinergic receptor excitation stimulant, may attenuate pulmonary inflammation and alleviate respiratory symptoms of ARDS patients in a clinical study (ChiCTR2100047403). In summary, we reveal a previously unrecognized MSC-mediated mechanism of CAP activation as the means by which MSCs alleviate ARDS-like syndrome, providing insight into the clinical translation of MSCs or CAP-related strategies for the treatment of patients with ARDS.

Metabolic and Cellular Compartments of Acetyl-CoA in the Healthy and Diseased Brain.

The human brain is characterised by the most diverse morphological, metabolic and functional structure among all body tissues. This is due to the existence of diverse neurons secreting various neurotransmitters and mutually modulating their own activity through thousands of pre- and postsynaptic interconnections in each neuron. Astroglial, microglial and oligodendroglial cells and neurons reciprocally regulate the metabolism of key energy substrates, thereby exerting several neuroprotective, neurotoxic and regulatory effects on neuronal viability and neurotransmitter functions. Maintenance of the pool of mitochondrial acetyl-CoA derived from glycolytic glucose metabolism is a key factor for neuronal survival. Thus, acetyl-CoA is regarded as a direct energy precursor through the TCA cycle and respiratory chain, thereby affecting brain cell viability. It is also used for hundreds of acetylation reactions, including N-acetyl aspartate synthesis in neuronal mitochondria, acetylcholine synthesis in cholinergic neurons, as well as divergent acetylations of several proteins, peptides, histones and low-molecular-weight species in all cellular compartments. Therefore, acetyl-CoA should be considered as the central point of metabolism maintaining equilibrium between anabolic and catabolic pathways in the brain. This review presents data supporting this thesis.

Comparative transcriptome analysis reveals the non-neuronal cholinergic system in the ovary of the oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae).

Acetylcholine (ACh), as a classical neurotransmitter, plays great roles in the nervous system. There is increasing evidence of its non-neuronal roles in regulating basic cell functions in vertebrates. However, knowledge about the non-neuronal cholinergic system in insects is scarce. A comparative transcriptome analysis was performed to investigate differences in the key molecular components of the cholinergic system between the head and ovary. The results showed that expression levels of most cholinergic system-related genes were higher in the head than in the ovary, and some cholinergic components were absent in the ovary. ACh contents ranged from 0.1 to 1.3μg mg-1 of wet weight during the development of the ovary, and weak acetylcholinesterase activity was also detected. Moreover, the ovary has a capacity for ACh synthesis. Bromoacetylcarnitine (BrACar), a specific carnitine acetyltransferase (CarAT) inhibitor, greatly inhibits ACh synthesis by 83.83% in ovary homogenates, but bromoacetylcholine (BrACh), a specific choline acetyltransferase (ChAT) inhibitor, has no effect on ACh synthesis in the ovary. These findings indicate that non-neuronal ACh in the ovary is only catalyzed by CarAT. This study reveals the existence of the non-neuronal cholinergic system in the ovary of M. separata, whose synthesis and release mechanisms are different from those of the head. These results provide novel insights into the non-neuronal cholinergic system in insects, and will be valuable in the discovery of new target genes and the future development of green pest control. © 2022 Society of Chemical Industry.

Effect of Calcium-Sensitive Receptor Agonist R568 on Gastric Motility and the Underlying Mechanism

Introduction: Calcium-sensitive receptor (CaSR) is expressed in the enteric nervous system of gastrointestinal tract. However, its role in the regulation of gastrointestinal motility has not yet been fully elucidated. We aimed to investigate the effect of the CaSR agonist – R568 on gastric motility and its potential mechanism. Methods: In vivo, R568 was given by gavage to explore gastric emptying with or without capsaicin which specifically blocks the function of vagal afferents; neurotransmitters synthetized in the myenteric plexus of the gastric corpus and antrum were analysed by ELISA and immunofluorescence staining; gastric muscle strips contraction recording and intracellular single unit firing recording were used to study the effect of R568 on muscle strips and myenteric interstitial cells of Cajal (ICCs) ex vitro. Results: Gastric emptying was inhibited by R568 in Kunming male mice, and capsaicin weakened this effect. The expression of c-fos-positive neurons increased in the nucleus tractus solitarius when R568 was treated. R568 decreased the expression of cholinergic neurons and reduced the synthesis of acetylcholine. Conversely, R568 increased the expression of nitrogenic neurons and enhanced the synthesis of nitric oxide in the myenteric plexus. Ex vitro results showed that R568 inhibited the contraction of the gastric antral muscle strip and suppressed the spontaneous firing activity of pacemaker ICCs. Conclusion: Activation of the gastrointestinal CaSR inhibited gastric motility in vivo and ex vitro. Transmitting nutrient signals to the brain through the vagal afferent nerve, modulating the cholinergic and nitrergic system in the enteric nervous system, and inhibiting activity of pacemaker ICCs in the myenteric plexus are involved in the mechanism of CaSR in gastric motility suppression.

Leptin Promotes Striatal Dopamine Release via Cholinergic Interneurons and Regionally Distinct Signaling Pathways.

Dopamine (DA) is a critical regulator of striatal network activity and is essential for motor activation and reward-associated behaviors. Previous work has shown that DA is influenced by the reward value of food, as well as by hormonal factors that reguate food intake and energy expenditure. Changes in striatal DA signaling also have been linked to aberrant eating patterns. Here we test the effect of leptin, an adipocyte-derived hormone involved in feeding and energy homeostasis regulation, on striatal DA release and uptake. Immunohistochemical evaluation identified leptin receptor (LepR) expression throughout mouse striatum, including on striatal cholinergic interneurons (ChIs) and their extensive processes. Using fast-scan cyclic voltammetry (FSCV), we found that leptin causes a concentration-dependent increase in evoked extra-cellular DA concentration ([DA]o) in dorsal striatum (dStr) and nucleus accumbens (NAc) core and shell in male mouse striatal slices, and also an increase in the rate of DA uptake. Further, we found that leptin increases ChI excitability, and that the enhancing effect of leptin on evoked [DA]o is lost when nicotinic acetylcholine (ACh) receptors are antagonized or when examined in striatal slices from mice lacking ACh synthesis. Evaluation of signaling pathways underlying leptin's action revealed a requirement for intracellular Ca2+, and the involvement of different downstream pathways in dStr and NAc core versus NAc shell. These results provide the first evidence for dynamic regulation of DA release and uptake by leptin within brain motor and reward pathways, and highlight the involvement of ChIs in this process.SIGNIFICANCE STATEMENT Given the importance of striatal dopamine (DA) in reward, motivation, motor behavior and food intake, identifying the actions of metabolic hormones on DA release in striatal subregions should provide new insight into factors that influence DA-dependent motivated behaviors. We find that one of these hormones, leptin, boosts striatal DA release through a process involving striatal cholinergic interneurons (ChIs) and nicotinic acetylcholine (ACh) receptors. Moreover, we find that the intracellular cascades downstream from leptin receptor (LepR) activation that lead to enhanced DA release differ among striatal subregions. Thus, we not only show that leptin regulates DA release, but also identify characteristics of this process that could be harnessed to alter pathologic eating behaviors.

Non-neuronal Cholinergic Muscarinic Acetylcholine Receptors in the Regulation of Immune Function

Immune cells such as T and B cells, monocytes and macrophages all express most of the cholinergic components of the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase (AChE). Because of its efficient cleavage by AChE, ACh synthesized and released from immune cells acts only locally in an autocrine and/or paracrine fashion at mAChRs and nAChRs on themselves and other immune cells located in close proximity, leading to modification of immune function. Immune cells generally express all five mAChR subtypes (M1-M5) and neuron type nAChR subunits α2-α7, α9, α10, β2-β4. The expression pattern and levels of mAChR subtypes and nAChR subunits vary depending on the tissue involved and its immunological status. Immunological activation of T cells via T-cell receptor-mediated pathways and cell adhesion molecules upregulates ChAT expression, which facilitates the synthesis and release of ACh. At present, α7 nAChRs expressed in macrophages are receiving much attention because they play a central role in anti-inflammatory cholinergic pathways. However, it now appears that through modification of cytokine synthesis, Gq/11-coupled mAChRs play a prominent role in regulation of T cell proliferation and differentiation and B cell immunoglobulin class switching. It is anticipated that greater understanding of Gq/11-coupled mAChRs on immune cells will provide an opportunity to develop new and effective treatments for immunological disorders.

The use of choline supplementation for the prevention of chemotherapy‐related cognitive impairments

The majority of patients who report chemotherapy‐related cognitive deficits are women. Many chemotherapeutic agents suppress ovarian function, decreasing circulating estrogen. Because estrogen regulates high affinity choline uptake (HACU) and HACU is the rate‐limiting step for acetylcholine (ACh) synthesis, chemotherapeutic agents may indirectly impair cholinergic mediated cognitive processes. ACh suppresses cytokine synthesis, inhibits inflammation and prevents tissue damage and cell death by activating α7 nicotinic ACh receptors (nAChRs), thus impaired HACU and ACh synthesis following chemotherapy may exacerbate the adverse effects of neuroinflammation caused by tumors and chemotherapeutic agents. Since increasing available choline can maintain cholinergic function when demand for the precursor is high, and because choline is a full agonist at α7 nAChRs, increasing dietary choline may protect ACh systems from degeneration by attenuating pro‐inflammatory processes.We have demonstrated that one administration of cyclophosphamide (CYP) and doxorubicin (DOX) impairs HACU in the striatum (STR) and hippocampus (HCC) of non tumor‐bearing (M‐) and tumor‐bearing MMTV‐PyVT (M+) female mice. Further, we have shown that the effect of one administration of CYP+DOX on HACU is prevented by placing mice on a 2% choline diet, and a 2% choline diet slows tumor growth in female M+ mice.The present study sought to determine whether a 2% choline diet could: 1) prevent the manifestation of spatial memory deficits resulting from repeated exposure to CYP (66.7mg/kg i.v.; ≍200mg/m2/wk) and DOX (6.7mg/kg i.v.; ≍20mg/m2/wk); 2) maintain HACU in the frontal cortex (CTX), STR and HCC of mice following 4 weekly administrations of CYP+DOX; 3) prevent reductions in circulating E2 consequent to repeated CYP+DOX exposure; 4) attenuate elevations of circulating cytokines induced by repeated CYP+DOX exposure and/or the presence of tumors; and 5) suppress tumor growth and enhance the antineoplastic effects of CYP+DOX treatment in female M+ mice.Results indicate that M‐ and M+ mice exposed to CYP+DOX exhibit deficits in spatial memory 10‐12 days following 4 weekly injections of CYP+DOX and 5 weeks following the final administration. HACU was reduced in the STR and HCC following a single exposure to CYP+DOX and this effect persisted in the STR. However, reduced HACU in the HCC was transient. Supplementation with a 2% choline diet prevented the manifestation of spatial memory deficits in M‐ and M+ mice and HACU in these mice did not differ from controls, suggesting that impaired HACU mediated the deficit in spatial memory. Although we did not observe an anticipated reduction in circulating estradiol due to CYP+DOX exposure, the regular appearance of proestrous was disrupted in groups exposed to either CYP+DOX or 2% choline diet, suggestive of amenorrhea. Circulating concentrations of cytokines, including G‐CSF, IL6, IP10, MCP1 and RANTES, were altered by the presence of tumors as well as the administration of CYP+DOX. 2% choline significantly reduced circulating concentrations of IL‐5, a cytokine associated with poor prognosis and metastasis, and total tumor volume was lower in mice placed on 2% choline diet when compared to tumor bearing mice on standard diet.

Evidence for Cholinergic Collateral Projections between Sympathetic Neurons in the Murine Stellate Ganglia

Postganglionic neurons of the stellate ganglia (SG) are an integral component of sympathetic regulation of the heart. SG neurons receive preganglionic cholinergic inputs via T‐2 and send noradrenergic projections to the heart via the inferior cardiac nerve (iCN) to influence cardiac activity. SG neurons are thought to receive simple monosynaptic inputs from preganglionic neurons, but early studies have hinted that cholinergic collateral projections may be present in the SG. In culture, SG neurons form noradrenergic and cholinergic synapses between one another, suggesting that SG neurons express dual neurochemical phenotypes. This study tests the hypothesis that cholinergic collateral projections are present between sympathetic neurons of the intact SG in mice.Whole‐cell patch clamp recordings were made of intact SG neurons from male (N=8) and female (N=6) ChATTH+CreERT2/lox mice with and without tamoxifen treatment (N=7 ChAT KO mice and N=7 control mice, respectively). Tamoxifen treatment in ChATTH+CreERT2/lox mice induced the deletion of choline acetyltransferase (ChAT) in tyrosine hydroxylase positive (i.e. noradrenergic) cells. This strategy blocks putative acetylcholine synthesis in SG neurons while leaving preganglionic cholinergic synapses intact. To search for collaterals, the preganglionic nerve trunk, T‐2, was electrically stimulated to evoke excitatory postsynaptic currents (eEPSCs). The amplitude and jitter (i.e. variability in latency) of eEPSCs were examined. Additionally, the postganglionic nerve trunk, the iCN, was electrically stimulated to evoke retrograde action currents (rAC) and the presence of evoked synaptic currents was assessed. The amplitude and frequency of spontaneous EPSCs (sEPSCs) were also examined.ChAT KO significantly reduced the frequency of sEPSCs compared to control mice (1.21±0.559events/s vs. 0.13±0.048events/s, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) while the amplitude was unaffected (74.4±26.10pA vs. 39.4±10.46pA, P>0.05 from Student’s unpaired t‐test, N=5‐6 cells). This suggests that ChAT KO reduces the presynaptic activity of postganglionic noradrenergic neurons within the SG. In control mice, stimulation of T‐2 evoked high jitter eEPSCs, which was significantly reduced in ChAT KO mice (327.1±92.14μs vs. 39.2±15.69μs, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) Additionally, the proportion of neurons that exhibited evoked synaptic currents following stimulation of the iCN was significantly reduced in ChAT KO mice (3/4 cells vs. 1/9 cells, P<0.05 from χ² test).Chemical transmission at additional synapses accounts for the high jitter events observed in disynaptic pathways. High jitter responses following T‐2 stimulation and evoked synaptic responses after iCN stimulation in control neurons are both consistent with the collateral hypothesis. The loss of high jitter responses and evoked synaptic currents in ChAT KO mice also suggests these collateral projections are cholinergic. This suggests that collateral synaptic activity contributes to the increased frequency of sEPSCs in control mice. Understanding SG neurocircuitry is critical in deciphering changes in sympathetic activity in many pathophysiological conditions such as myocardial infarction and heart failure and may identify novel therapeutic targets for the treatment of autonomic imbalance.

Immunocytochemical Localization of Enzymes Involved in Dopamine, Serotonin, and Acetylcholine Synthesis in the Optic Neuropils and Neuroendocrine System of Eyestalks of Paralithodes camtschaticus.

Identifying the neurotransmitters secreted by specific neurons in crustacean eyestalks is crucial to understanding their physiological roles. Here, we combined immunocytochemistry with confocal microscopy and identified the neurotransmitters dopamine (DA), serotonin (5-HT), and acetylcholine (ACh) in the optic neuropils and X-organ sinus gland (XO-SG) complex of the eyestalks of Paralithodes camtschaticus (red king crab). The distribution of Ach neurons was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared with that of DA neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. We detected 5-HT, TH, and ChAT in columnar, amacrine, and tangential neurons in the optic neuropils and established the presence of immunoreactive fibers and neurons in the terminal medulla in the XO region of the lateral protocerebrum. Additionally, we detected ChAT and 5-HT in the endogenous cells of the SG of P. camtschaticus for the first time. Furthermore, localization of 5-HT- and ChAT-positive cells in the SG indicated that these neurotransmitters locally modulate the secretion of neurohormones that are synthesized in the XO. These findings establish the presence of several neurotransmitters in the XO-SG complex of P. camtschaticus.

The nonneuronal cholinergic system in the colon: A comprehensive review

Acetylcholine (ACh) is found not only in cholinergic nerve termini but also in the nonneuronal cholinergic system (NNCS). ACh is released from cholinergic nerves by vesicular ACh transporter (VAChT), but ACh release from the NNCS is mediated by organic cation transporter (OCT). Recent studies have suggested that components of the NNCS are located in intestinal epithelial cells (IECs), crypt-villus organoids, immune cells, intestinal stem cells (ISCs), and vascular endothelial cells (VECs). When ACh enters the interstitial space, its self-modulation or effects on adjacent tissues are part of the range of its biological functions. This review focuses on the current understanding of the mechanisms of ACh synthesis and release in the NNCS. Furthermore, studies on ACh functions in colonic disorders suggest that ACh from the NNCS contributes to immune regulation, IEC and VEC repair, ISC differentiation, colonic movement, and colonic tumor development. As indicated by the features of some colonic disorders, ACh and the NNCS have positive and negative effects on these disorders. Furthermore, the NNCS is located in multiple colonic organs, and the specific effects and cross-talk involving ACh from the NNCS in different colonic tissues are explored.

Duodenal infusion of soy protein hydrolysate activates cAMP signaling and hypothalamic neurotransmitter synthesis in pigs.

Neurotransmitters in the brain are important for cognition and memory. As bioactive substrates, whether increased soy protein levels in pigs can promote hypothalamic neurotransmitter synthesis remains unclear. The effect of increased soy protein hydrolysate (SPH) levels in the small intestine of pigs on neurotransmitter precursor supply, hypothalamic neurotransmitter synthesis and underlying molecular processes was investigated by using sixteen pigs (35.2 ± 0.3 kg) infused either with SPH (70 g day-1) or sterile saline (control) twice daily for 15 days via a duodenal fistula. It demonstrated that SPH infusion increased the expression of the neutral amino acid transporter B0AT1 in the jejunal mucosa, the serum tyrosine/large neutral amino acid ratio, the concentrations of serum tyrosine, hypothalamic tyrosine, dopamine and brain-derived neurotrophic factor (BDNF) (P < 0.05). It also increased the jejunal and serum choline, hypothalamic choline and acetylcholine levels (P < 0.05). Hypothalamic transcriptome revealed that differential genes were significantly enriched in the cholinergic synapse, dopaminergic synapse and cyclic adenosine monophosphate (cAMP) signalling pathways, and that the expression of key enzyme genes in the synthesis of acetylcholine and dopamine and dopamine receptors 1 (DRD1) was upregulated by SPH (P < 0.05). Western blotting showed that SPH infusion activated the hypothalamic cAMP signalling pathways. Overall, SPH infusion promoted the synthesis of hypothalamic dopamine and acetylcholine, and the synthesised dopamine promoted BDNF production most likely through the activation of the cAMP signalling pathways by the DRD1.

The role of vascular injury within the conditions of choline deficiency in rats with scopolamine-induced alzheimer's type dementia

Background. The relationship between choline deficiency and vascular dysfunction continues to be relevant in the study of Alzheimer's disease. Objective. To study the morphological characteristics of vascular injury within the conditions of choline deficiency in rats with scopolamine-induced Alzheimer's type dementia. Methods. The experiment was performed on 48 WAG population male rats weighing 180-230 gr. Rats from groups Scop-14, Scop-14-SC, Scop-28, Scop-28-SC were injected intraperitoneally with scopolamine (Scop) butylbromide at a dosage of 1 mg/kg of body mass during 14 and 28 days and intravenously with mesenchymal stem cells (SC) at a single dosage of 500000 cells per 1 rat. Control animals (gr.C) were injected with 0.9% sodium chloride. Brain slides were stained with Congo-red and gallocyanine-chromium alum according to Einarson's method for total nucleic acids. The VEGF, E-cadherin expression was immunohistochemically determined in the brain cells cytoplasm. Results. The congophilic staining of the arteries walls, a decrease in endothelial cells with low the E-cadherin expression and an increase in the number of pericytes in the capillary wall was observed in the experimental groups. In gr.Scop-28 VEGF expression in endothelial cells, hippocampal neurons was greater than in gr.Scop-14. It indicated more intensive activation of angiogenesis and acetylcholine synthesis with correspondingly more pronounced vascular damage and choline deficiency. The cytoplasm of cortical neurons was diffusely labeled with VEGF antibodies in response to hypoxia, but the level of expression was almost no different from that in gr.C. In all groups, the optical density of the neuropile of the large hemispheres according to Einarson’s staining was reduced, i.e., the level of RNA in the neuronal processes was reduced. The introduction of stem cells restored the capillary wall due to young endothelial cells, reduced the VEFG synthesis in all studied cells and increased the RNA content in neuronal processes. Conclusion. The relationship between choline deficiency, neuronal process loss and vascular damage has been found. The blood vessels self-repair was occurred by substitution, after the stem cells introduction - by restitution.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis.

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high‐affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Reduction of acetylcholine in the hippocampus of hippocampal cholinergic neurostimulating peptide precursor protein knockout mice

The cholinergic efferent network from the medial septal nucleus to the hippocampus plays an important role in learning and memory processes. This cholinergic projection can generate theta oscillations in the hippocampus to encode novel information. Hippocampal cholinergic neurostimulating peptide (HCNP), which induces acetylcholine (Ach) synthesis in the medial septal nuclei of an explant culture system, was purified from the soluble fraction of postnatal rat hippocampus. HCNP is processed from the N-terminal region of a 186-amino acid, 21-kDa HCNP precursor protein, also known as Raf kinase inhibitory protein and phosphatidylethanolamine-binding protein 1. Here, we confirmed direct reduction of Ach release in the hippocampus of freely moving HCNP-pp knockout mice under an arousal state by the microdialysis method. The levels of vesicular acetylcholine transporter were also decreased in the hippocampus of these mice in comparison with those in control mice, suggesting there was decreased incorporation of Ach into the synaptic vesicle. These results potently indicate that HCNP may be a cholinergic regulator in the septo-hippocampal network.

Immunohistochemical Expression of Choline Acetyltransferase and Catecholamine-Synthesizing Enzymes in Head-and-Neck and Thoracoabdominal Paragangliomas and Pheochromocytomas.

Paragangliomas (PGLs) are neural-crest-derived, non-epithelial neuroendocrine tumors distributed along the parasympathetic and sympathetic nerves. Head-and-neck PGLs (HNPGLs) have been recognized as nonchromaffin, nonfunctional, parasympathetic tumors. By contrast, thoracoabdominal paragangliomas and pheochromocytomas (PPGLs) are chromaffin, functional, sympathetic tumors. Although HNPGLs and PPGLs have the same histological structure, the zellballen pattern, composed of chief and sustentacular cells surrounded by abundant capillaries, the pathobiological differences between these types of PGLs remain unclarified. To determine the phenotypic features of these PGLs, we performed an immunohistochemical study using specific antibodies against choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, and enzymes for the catecholamine-synthesis, tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH), in 34 HNPGLs from 31 patients, 12 thoracoabdominal PGLs from 12 patients, and 26 pheochromocytomas from 22 patients. The expression of ChAT, TH, and DBH was 100%, 23%, and 10% in the HNPGLs; 12%, 100%, and 100% in the pheochromocytomas; and 25%, 67%, and 100% in the thoracoabdominal PGLs, respectively. These results designate HNPGLs as acetylcholine-producing parasympathetic tumors, in contrast to PPGLs being catecholamine-producing tumors. The other most frequently used neuroendocrine markers are synaptophysin and chromogranin A expressed 100% and 80%, respectively, and synaptophysin was superior to chromogranin A in HNPGLs. This is the first report of HNPGLs being acetylcholine-producing tumors. Immunohistochemistry of ChAT could be greatly useful for pathologic diagnosis of HNPGL. Whether measurement of acetylcholine levels in the blood or urine could be a tumor marker of HNPGLs should be investigated soon.