High-affinity Choline Transporter CHT1 Research Articles

Competitive inhibition of the high-affinity choline transporter by tetrahydropyrimidine anthelmintics

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 μM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.

Dietary choline deficiency aggravated the intestinal apoptosis in association with the MAPK signalling pathways of juvenile grass carp (Ctenopharyngodon idella)

To investigate the effects and potential mechanisms of apoptosis in the intestines of juvenile grass carp (Ctenopharyngodon idella) for dietary choline, fish were fed different choline-containing diets (142.2, 407.4, 821.6, 1215.8, 1589.3, and 1996.6 mg kg−1) for 70 days. Results revealed that dietary choline deficiency (1) decreased the contents of choline, phosphatidylcholine, betaine, and acetylcholine (not in distal intestine [DI]) in grass crap intestines; down-regulated the mRNA abundances of choline transportations, such as high-affinity choline transporter CHT1 (not in proximal intestine[PI] and DI), choline transporter-like proteins such as CTL-1, CTL-2, CTL-4 (not in DI), and CTL-5 in grass crap intestines; (2) enhanced the degree of DNA fragments. Moreover, it up-regulated the activities of caspase-3, −8, and-9; reduced mRNA levels of caspase-3, −7, −8 (not in DI), and − 9, Fas ligand (FASL) (not in PI), and apoptotic protease activating factor-1(Apaf1), as well as the protein level of cytochrome c (Cty c). Dietary choline deficiency also down-regulated myeloid cell leukemia-1 (Mcl-1) (not in PI), B-cell lymphoma-2 (Bcl-2), and the inhibitor of apoptosis protein (IAP) mRNA levels in grass carp intestines, suggesting that choline inhibited cell apoptosis in fish intestines; (3) increased p38MAPK and JNK(not in mid intestine [MI]) mRNA level, as well as p-p38MAPK and T-p38MAPK protein levels in the PI and the ratio of p-p38MAPK/T-p38MAPK in the MI and DI, indicating that choline deficiency aggravated cell apoptosis in association with the MAPK signalling pathway in fish intestines; In conclusion, choline insufficiency promotion of intestinal apoptosis might be related to MAPK signalling pathways in fish. Moreover, the requirements of choline for resist intestinal apoptosis indices (caspase-8 and capase-9 activities) of grass carp (9.28–108.97 g) were calculated to be (1162.13–1780.93) mg kg−1 diet, respectively.

Upregulation of the high-affinity choline transporter in colon relieves stress-induced hyperalgesia.

BackgroundIrritable bowel syndrome (IBS) is a common disease with hyperalgesia, the mechanisms of which remain elusive. The cholinergic system is known to be involved in pain inhibitory pathways in multiple diseases, and its involvement in IBS is unknown.ObjectiveWe aimed to determine whether high-affinity choline transporter CHT1, a major determinant of the cholinergic signaling capacity, is involved in regulating intestinal sensations associated with stress-induced visceral pain.Materials and methodsAn IBS rat model was established by chronic water avoidance stress (WAS). Colonic pathologic alterations were detected by H&E staining. Visceral sensations were determined by scoring the abdominal withdrawal reflex (AWR) and visceromotor response (VMR) magnitude of the electromyogram in response to colorectal distension (CRD). Abdominal mechanical hyperalgesia was assessed by counting the number of withdrawal events evoked by applying von Frey filaments. Real-time PCR, Western blot, and immunostaining were performed to identify CHT1 expression in the colon. Acetylcholine (ACh) secretion was determined by ELISA. Effects of MKC-231, a choline uptake enhancer, on visceral pain were examined.ResultsAfter 10 days of WAS exposure, AWR score and VMR magnitude in response to CRD were significantly enhanced and the number of withdrawal events was elevated. Protein and mRNA levels of CHT1 were considerably increased in the colon after WAS. CHT1 upregulation in the WAS-exposed group was largely abolished by ammonium pyrrolidinedithiocarbamate. The density of CHT1-positive intramuscular cells and enteric neurons in the myenteric plexus was enhanced in WAS-exposed rats. Pharmacologic enhancement of CHT1 activity by MKC-231 gavage could relieve the visceral pain of WAS rats by upregulating CHT1 protein expression and enhancing ACh production.ConclusionCHT1 may exert an antinociceptive effect in stress-induced visceral pain by modulating ACh synthesis through nuclear factor kappa B signaling. MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.

Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis

BackgroundThe mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. MethodsCP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. ResultsSigns of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. ConclusionWe report for the first time that CHT1 may be involved in pain modulation in CP, as it plays an important role in pain inhibition. Increased CHT1 activity or the up-regulation of its expression may be used to treat pain in patients with CP.

Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon

Luminal short-chain fatty acids (SCFAs) influence gut physiological function via SCFA receptors and transporters. The contribution of an SCFA receptor, free fatty acid receptor (FFA)3, to the enteric nervous system is unknown. FFA3 is expressed in enteric cholinergic neurons. Activation of neural FFA3 suppresses Cl(-) secretion induced by nicotinic ACh receptor activation via a Gi/o pathway. Neural FFA3 may have an anti-secretory function by modulating cholinergic neural reflexes in the enteric nervous system. The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl(-) secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13μm. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the Gi/o pathway. Luminal propionate induced Cl(-) secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

Neural FFA3 Negatively Regulates Cholinergic Secretory Function in Rat Proximal Colon

The lumen of the proximal colon is continuously exposed to high concentrations of short‐chain fatty acids (SCFAs). Although propionate evokes transient anion secretion via neural and non‐neural cholinergic pathways in the colon, the involvement of FFA3 (GPR41) and its absorptive pathway have not been clarified. We investigated the expression of FFA3 and its contribution towards propionate‐ and carbachol (CCh)‐induced secretory responses in rat proximal colon. FFA3 immunolocalized to the enteroendocrine cells and to the enteric neural plexuses. Most of the FFA3‐immunoreactive nerve fibers were cholinergic, based on staining with ChAT, a vesicular ACh transporter, and the high affinity choline transporter CHT1. In Ussing chambered proximal colon, luminal propionate induced anion secretion in fasted, but not fed conditions. TTX or atropine reduced the response to propionate by 60% or 94%, respectively. In the presence of indomethacin, the non‐substrate sodium‐dependent monocarboxylate transporter (SMCT)1 inhibitor ibuprofen or fenoprofen abolished the response to propionate, without inhibition on CCh‐evoked secretion. CCh‐induced secretion was decreased by TTX or serosal propionate by 40%. Serosal application of the FFA3 agonist decreased the response to propionate and CCh, indicating that FFA3 negatively regulates cholinergic neural reflexes. These results suggest that luminal propionate‐induced anion secretion is dependent on SMCT1‐mediated SCFA absorption, which in turn varies cholinergic nerve activity.Supported by VA Merit Review, NIH R01 DK54221

Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter

The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na(+)/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na(+)/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.

The high-affinity choline transporter CHT1 is regulated by the ubiquitin ligase Nedd4-2

The high-affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons, constitutes a rate-limiting step for acetylcholine synthesis. We have found that the exogenous ubiquitin ligase Nedd4-2 interacts with CHT1 expressed in HEK293 cells decreasing the amount of cell surface CHT1 by approximately 40%, and that small interfering RNA for endogenous Nedd4-2 enhances the choline uptake activity by CHT1 in HEK293 cells. These results indicate that Nedd4-2-mediated ubiquitination regulates the cell surface expression of CHT1 in cultured cells and suggest a possibility that treatments or drugs which inhibit the interaction between CHT1 and Nedd4-2 might be useful for diseases involving decrease in acetylcholine level such as Alzheimer's disease.

Substrate-Induced Internalization of the High-Affinity Choline Transporter

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

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Role of Acetylcholine and Muscarinic Receptors in Serotonin-Induced Bronchoconstriction in the Mouse

For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice

The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy.

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Expression of the high-affinity choline transporter CHT1 in epithelia

Uptake of choline by the high-affinity choline transporter CHT1 is the rate-limiting step in neuronal acetylcholine (ACh) synthesis. Here, we investigated by RT-PCR, in-situ hybridisation, immunohistochemistry, and Western blotting whether CHT1 is also expressed in cholinergic epithelia. CHT1-mRNA and -protein were detected in keratinocytes of human skin, rat skin and tongue, the human keratinocyte cell line HaCaT, and the ciliated cells of the rat tracheal epithelium. Immunohistochemically, CHT1 was predominantly localized to the epithelial cell membranes, in case of ciliated tracheal cells it was restricted to the apical membrane. This is the first study to demonstrate the expression of CHT1 in non-neuronal cells. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favour of ACh synthesis being fuelled by choline uptake via CHT1 in these epithelia.

The neuronal choline transporter CHT1 is regulated by immunosuppressor-sensitive pathways.

The immunosuppressor cyclosporin A inhibits the peptidyl-prolyl-cis/trans-isomerase activity of cyclophilins and the resulting complex inhibits the phosphatase activity of calcineurin. Both enzymes were detected in peripheral nerve endings isolated from the electric organ of Torpedo and shown to be affected by 10 micro m cyclosporin A. Among the cholinergic properties studied, choline uptake was specifically inhibited by cyclosporin A to a maximum of 40%. Cyclosporin A decreased the rate of choline transport but not the binding of the non-transportable choline analogue hemicholinium-3, indicating that the number of membrane transporters was not affected. Through the use of two other immunosuppressors, FK506, which also inhibits calcineurin, and rapamycin, which does not, two different mechanisms of choline uptake inhibition were uncovered. FK506 inhibited the rate of choline transport, whereas rapamycin diminished the affinity for choline. The Torpedo homologue of the high affinity choline transporter CHT1 was cloned and its activity was reconstituted in Xenopus oocytes. Choline uptake by oocytes expressing tCHT1 was inhibited by all three immunosuppressors and also by microinjection of the specific calcineurin autoinhibitory domain A457-481, indicating that the phosphatase calcineurin regulates CHT1 activity and could be the common target of cyclosporin and FK506. Rapamycin, which changed the affinity of the transporter, may have acted through an immunophilin on the isomerization of critical prolines that are found in the tCHT1 sequence.

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na +-independent and shows a Michaelis–Menten constant of approx. 20 μM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 μM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na +-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na +-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.