The choline acetyltransferase of human placenta.
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ABSTRACT:
SUBMITTER: Morris D
PROVIDER: S-EPMC1264916 | biostudies-other | 1966 Mar
REPOSITORIES: biostudies-other
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Project description:IntroductionThe developing fetus relies on the maternal blood supply to provide the choline it requires for making membrane lipids, synthesizing acetylcholine, and performing important methylation reactions. It is vital, therefore, that the placenta is efficient at transporting choline from the maternal to the fetal circulation. Although choline transporters have been found in term placenta samples, little is known about what cell types express specific choline transporters and how expression of the transporters may change over gestation. The objective of this study was to characterize choline transporter expression levels and localization in the human placenta throughout placental development.MethodsWe analyzed CTL1 and -2 expression over gestation in human placental biopsies from 6 to 40 weeks gestation (n = 6-10 per gestational window) by immunoblot analysis. To determine the cellular expression pattern of the choline transporters throughout gestation, immunofluorescence analysis was then performed.ResultsBoth CTL1 and CTL2 were expressed in the chorionic villi from 6 weeks gestation to term. Labor did not alter expression levels of either transporter. CTL1 localized to the syncytial trophoblasts and the endothelium of the fetal vasculature within the chorionic villous structure. CTL2 localized mainly to the stroma early in gestation and by the second trimester co-localized with CTL1 at the fetal vasculature.DiscussionThe differential expression pattern of CTL1 and CTL2 suggests that CTL1 is the key transporter involved in choline transport from maternal circulation and both transporters are likely involved in stromal and endothelial cell choline transport.
| S-EPMC4801320 | biostudies-literature
Project description:Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.
| S-EPMC3196808 | biostudies-literature
Project description:Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.
| S-EPMC3037696 | biostudies-literature
Project description:1. Michaelis constants for human placental choline acetyltransferase were shown to be dependent on the concentration of the second substrate present. The primary plots indicate a sequential rather than a Ping Pong mechanism and are of the same type with 300mm- and 500mm-sodium chloride. 2. Similar results have been obtained with rabbit brain choline acetyltransferase. 3. Product inhibition of the forward reaction has been studied. CoA inhibits competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibits competitively with respect to choline and non-competitively with respect to acetyl-CoA. No inhibition is given by acetylcholine when the enzyme is saturated with choline. 4. It is concluded that human placental choline acetyltransferase has an ordered mechanism of the Theorell-Chance type.
| S-EPMC1178191 | biostudies-other
Project description:Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been determined. We previously showed that CD4+ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals. Here we show that these CD4+CD44hiCD62Llo T helper cells by gene expression are a distinct T-cell population defined by ChAT (CD4 TChAT). Mice lacking ChAT expression in CD4+ cells have elevated arterial blood pressure, compared to littermate controls. Jurkat T cells overexpressing ChAT (JTChAT) decreased blood pressure when infused into mice. Co-incubation of JTChAT and endothelial cells increased endothelial cell levels of phosphorylated endothelial nitric oxide synthase, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasorelaxant nitric oxide. The isolation and characterization of CD4 TChAT cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation.
| S-EPMC5513182 | biostudies-literature
Project description:Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.
| S-EPMC6476985 | biostudies-literature
Project description:Alzheimer disease and related dementias are major challenges, demanding urgent needs for earliest possible diagnosis to optimize the success rate in finding effective therapeutic interventions. Mounting solid scientific premises point at the core acetylcholine-biosynthesizing cholinergic enzyme, ChAT as a legitimate in vivo target for developing positron emission tomography biomarker for early diagnosis and/or monitoring therapeutic responses in the neurodegenerative dementias. Up-to-date, no PET tracer ligands for ChAT are available. Here we report for the first time a novel hierarchical virtual screening approach on a commercial library of ~300,000 compounds, followed by in vitro screening of the hits by a new High-Throughput ChAT assay. We report detailed pharmacodynamic data for three identified selective novel ChAT ligands with IC50 and K i values ranging from ~7 to 26?µM. In addition, several novel selective inhibitors of the acetylcholine-degrading enzymes, AChE and BuChE were identified, with one of the compounds showing an IC50-value of ~6?µM for AChE. In conclusion, this report provides an excellent starting platform for designing and optimizing potent and selective ChAT ligands, with high potential as PET-imaging probe for early diagnosis of AD, and related dementias, such as Down's syndrome and Lewy body disorders.
| S-EPMC5701137 | biostudies-literature
Project description:Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.
| S-EPMC424412 | biostudies-literature
Project description:Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker L-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.
| S-EPMC8529785 | biostudies-literature
Project description:We have identified a zebrafish mutant line, bajan, in which compromised motility and fatigue result from a point mutation in the gene coding choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. Although the mutation predicts loss of ChAT function, bajan inexplicably retains low levels of neuromuscular transmission. We exploited this residual activity and determined the consequences for synaptic function. The attenuated synaptic responses were a direct consequence of a decrease in both resting mean quantal size and quantal content. To replicate behavioral fatigue in swimming, motorneurons were stimulated at high frequencies. A prominent reduction in quantal content, reflecting vesicle depletion, was coincident with a small additional reduction in quantal size. In humans, defective ChAT leads to episodic apnea, a form of congenital myasthenic syndrome characterized by use-dependent fatigue. In contrast to bajan, however, afflicted individuals exhibit a normal resting quantal size and quantal content. The fatigue in humans results from a pronounced long-lasting drop in quantal size with little or no change in quantal content. These differences have important implications for interpreting fatigue as well as on understanding the impact of ACh availability on vesicle filling and recycling.
| S-EPMC2576202 | biostudies-literature