Inhibition Of Choline Transport Research Articles

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition and inactivation of presynaptic cholinergic markers using redox-reactive choline analogs.

Inhibition and inactivation of two presynaptic cholinergic "markers", choline acetyltransferase and high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive catechol tethered to a quaternary ammonium group. Two quaternary ammonium alkyl-substituted catechols, 3[(trimethylammonio)methyl]catechol (TMC, 1) and N,N-dimethylepinephrine (catecholine, 2) were shown to bind weakly and noncompetitively to bovine choline acetyltransferase yet inactivated the enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these catechols. Both agents also inhibited high-affinity choline uptake. The time course of TMC and catecholine spontaneous oxidation-dependent inactivation of high affinity choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable choline transport in synaptosomes. When compared with inhibition of uptake of other neurotransmitters, it was shown that catecholine demonstrated more selectivity than TMC toward inhibition of choline transport. Km (microM) and Vmax (pmol/min per mg of protein) were measured for high affinity transport of choline, dopamine, and serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for catecholine and TMC, calculated from experimentally determined IC50's, were for catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for choline, dopamine, and serotonin transport, respectively. In vivo studies using catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus, catecholine represents a lead compound in a potential series of redox-reactive choline analogs, which may become useful irreversible antagonists of the critical cholinergic macromolecular targets underlying cholinergic hypofunction in disorders such as Alzheimer's disease.

Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.

Inhibitors of choline transport in alveolar type II epithelial cells.

Isolated alveolar type II epithelial cells (granular pneumocytes) from rat lung accumulate free choline against a concentration gradient by an energy-dependent saturable transport process with apparent Km approximately 18 microM. In order to evaluate the structural requirements for choline transport by these cells, the inhibition of the initial rate of cellular uptake of [3H]choline (5 microM) by its analogue was measured. There was no significant inhibition of substrate uptake by analogues lacking an amino group while the presence of a quaternary nitrogen was most effective. N,N'-dimethylethanolamine (apparent Ki, 7 microM) and n-decylcholine (apparent Ki, 0.5 microM) were potent competitive inhibitors of choline transport. Substitution of the hydroxyl group in choline greatly diminished the inhibitory effect; fluorocholine, thiocholine, betaine, and betaine aldehyde showed little or no inhibition. This requirement for a hydroxyl group raises the possibility of hydrogen bonding of choline with the transport protein. The choline transport system in granular pneumocytes appears to differ from that in synaptosomes by the lower affinity of the carrier for substrate and for hemicholinium-3 and from that in erythrocytes by the role of the hydroxyl in the substrate molecule. The availability of inhibitory analogues for choline transport will facilitate isolation and study of the granular pneumocyte choline transport protein.

Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca2+

The major route of phosphatidylcholine (Ptd-choline) biosynthesis in mammalian cells is the CDP-choline pathway which involves stepwise conversion of choline to phosphocholine (P-choline), cytidine diphosphate choline (CDP-choline), and Ptd-choline. Our previous studies with electropermeabilized (EP) rat glioma (C6) cells have indicated that the intermediates of this pathway are not freely diffusible in the cell but are channeled toward synthesis of Ptd-choline (George, T.P., Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F. B. St.C., and Spence, M.W. (1989) Biochim. Biophys. Acta 1004, 283-291). In this study, Ca(2+)-[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers were used to investigate the role of intracellular free Ca2+ levels in functional organization of this pathway in EP glioma cells. In EP cells reduction of free Ca2+ in the medium from 1.8 mM to less than 200 nM resulted in 2-3-fold stimulation of exogenous [3H]choline and [14C]P-choline incorporation into Ptd-choline whereas incorporation of exogenous CDP-[14C]choline was augmented 100-fold; there was no uptake or incorporation of labeled P-choline or CDP-choline in intact cells. In EP cells incubated at 1.8 mM Ca2+ the water-soluble products of choline metabolism (choline, P-choline, CDP-choline, and glycerophosphocholine) were retained at 37 degrees C; in contrast, in the presence of 100 nM Ca2+ there was uniform leakage of these metabolites. Experiments with hemicholinium-3, an inhibitor of choline transport, and EP cells at 100 nM Ca2+ show that linkage of choline transport and Ptd-choline biosynthesis is also dependent on Ca2+. These results suggest that channeling of intermediates in the CDP-choline pathway of Ptd-choline biosynthesis in glioma cells is mediated by intracellular Ca2+ levels that may coordinately regulate the steps involved in conversion of choline to Ptd-choline.

Positive and Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Cortical Prisms Incubated Under “Resting”Conditions

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low-potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under "resting" conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium-3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium-3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of choline transport in erythrocytes by n-alkanols

The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal / mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7–1.9. The mechanism of inhibition is noncompetitive, and partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.

Choline transport specificity in animal cells and tissues

1. 1. Beta carbolines inhibit choline transport in rat brain. 2. 2. The aziridinium ring on the nitrogen of mustard analogs of choline causes irreversible binding to the carrier in rat brain. 3. 3. The uptake system in rat brain is stereoselective, requires a quaternary nitrogen, and prefers analogs with a nitrogen-oxygen distance of about 3.26 Å. 4. 4. In mouse brain troxonium derivatives inhibit choline transport. 5. 5. In cuttlefish optic lobes and torpedo electric organ pyrene derivatives potently inhibit choline transport. 6. 6. In guinea pig placenta, the affinity of the choline carrier remains high even when this molecule lacks one or two methyl groups.

Non-specific effects of the putative cholinergic neurotoxin ethylcholine mustard aziridinium ion in the rat brain examined by autoradiography, immunocytochemistry and gel electrophoresis

Autoradiographic localisation of [ 3H]-ethylcholine mustard aziridinium ion (ECMA) after microinjection into the rat striatum has revealed intracellular sequestration of the toxin by glial and endothelial cells; fewer neuronal cells were labelled. Intrastriatal injection of 200 pmol ECMA caused severe cavitation of the tissue, extensive gliosis and permanent damage to myelinated structures, as revealed by immunocytochemical detection of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). These nonspecific effects are in addition to ECMA's irreversible action on the choline carrier associated with cholinergic neurons, and only marginally protected by concomitant administration of the reversible choline transport inhibitor hemicholinium-3. They may instead be attributed to the powerful alkylating action that ECMA has on tissue proteins, as shown by fluorography of synaptosomal proteins treated with [ 3H]ECMA and separated by SDS-PAGE.

Specificity of ethylcholine mustard aziridinium as an irreversible inhibitor of choline transport in cholinergic and noncholinergic tissue.

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal.

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.

Inhibitors of choline transport into Plasmodium-infected erythrocytes are effective antiplasmodial compounds in vitro

Inhibitors of choline transport into Plasmodium-infected erythrocytes are effective antiplasmodial compounds in vitro

Characterization of choline transport at maternal and fetal interfaces of the perfused guinea‐pig placenta.

Unidirectional influx and efflux of choline into the syncytiotrophoblast were investigated from both maternal and fetal circulations of the perfused guinea-pig placenta by using a single-circulation paired-tracer (extracellular reference and test substrate) dilution technique. Cellular uptake of [3H]choline at 0.05 mM was (mean percentage +/- S.E. of mean, n = 14 placentae) 51 +/- 2 and 49 +/- 2, on maternal and fetal sides, respectively. Kinetics of unidirectional influx (0.05-4.0 mM-choline) indicated the existence of saturable and non-saturable components on both sides: on maternal and fetal interfaces the Km (mM) values were respectively, 0.12 and 0.13, the Vmax (mumol min-1 g-1) values, 0.08 and 0.07 and the apparent linear transfer constants (min-1 g-1) 0.11 and 0.12. Efflux of [3H]choline from the placenta back into the ipsilateral circulation (backflux) was generally fast (20-60% in 5-6 min) and asymmetric with the fetal: maternal ratio usually above unity. Transplacental specific choline transfer in the dually perfused placenta, when observed, was small (less than 10% of the injected dose) following tracer injections in either direction based on the 5-6 min collection of the contralateral circulation (at 0.05 mM-choline). Placental retention of [3H]choline at the end of the 5-6 min period was about 25% of the injected dose when the tracers were injected from either circulation. Analogues of choline such as hemicholinium-3, thiamine, ethanolamine and N,N-dimethylethanolamine inhibited choline unidirectional influx, whereas betaine and acetate had no effect. The absence of the normal sodium gradient (perfusate sodium was replaced by Tris or by lithium) did not inhibit choline transport. The metabolic inhibitors dinitrophenol (1.0 mM) and potassium cyanide (1.0 mM) were essentially ineffective (up to 40 min perfusion). The sulphydryl reagent N-ethylmaleimide did not appear to inhibit the influx, in comparison with its effect on [3H]choline backflux which was greatly accelerated, resulting in a dramatic reduction in placental net uptake of the label. Our findings show that choline transport into the placenta is a rapid carrier-mediated process occurring at both maternal and fetal sides of the trophoblast, at physiological blood concentrations. This cellular uptake is possibly related to the synthesis of acetylcholine, which is known to occur in human placental tissue. Specific transplacental transfer of choline was a very slow process under the conditions of our experiments and this contrasted with the observed fast and high uptake into the trophoblast.

Choline transport across a carbon tetrachloride phase containing a chloroform-methanol extract of brain

The presence of a chloroform-methanol extract of cat brain in a carbon tetrachloride phase separating two aqueous phases resulted in an increased passage of [ 3H]choline across the organic phase which was inhibited by the choline transport inhibitor hemicholinium-3 and by high concentrations of non-radioactive choline. In the absence of cat brain extract, [ 3H]choline passage across carbon tetrachloride was neither inhibited by hemicholinium-3, nor by non-radioactive choline.

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism.

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.

Do raised brain aluminium levels in Alzheimer's dementia contribute to cholinergic neuronal deficits?

Raised aluminium levels have been found in brains of patients with Alzheimer's dementia (1,2), a disease in which reductions have been reported in various parameters of presynaptic cholinergic nerve function, including choline uptake, acetylcholine synthesis and choline acetyltransferase activity (3). Aluminium has been found to inhibit choline transport by isolated rat brain nerve endings (4) and human erythrocytes (5), and also to cause an encephalopathy in rabbits with neurofibrillary tangles and reduced neuronal choline acetyltransferase activity (6). It is therefore hypothesised that raised brain aluminium levels in Alzheimer's dementia may contribute to the cholinergic neuronal deficits in this disease. If this is the case, then aluminium chelating agents may be of value in its treatment.

The effects of Al 3+, Cd 2+ and Mn 2- on human erythrocyte choline transport

The effects of Al 3+, Cd 2+ and Mn 2+ on human erythrocyte choline transport, Na-K-ATPase, Ca-Mg-ATPase and intracellular K + levels were examined. The concentrations used were below the levels which caused significant haemolysis (⩽300 μM). All three cations inhibited concentrative choline accumulation over 3 hr [ ic 50 values at 1 μM choline were 35 μm (AlCl 3), 250 μM (CdCl 2) and 300 μm (MnCl 2)] but at the concentrations tested, none decreased initial rates of choline uptake. The effects of Cd 2− and Mn 2− (but not Al 3+) on choline accumulation were reversed by removing the cations from the extracellular medium by washing. All three cations also inhibited efflux of choline. at 1 μM choline, 30% inhibition being produced by 33 μM AlCl 3, 81μM CdCl 2 and 111 μM MnCl 2. At subhaemolytic concentrations, only CdCl 2 inhibited Na-K-ATPase, ( ic 50 = 147 μ M) and none of the cations significantly inhibited Ca-Mg-ATPase. Intracellular K − levels were only reduced by the highest concentration of AlCl 3 used (100 μM). These results suggest that inhibition of choline accumulation and efflux in erythrocytes by Al 3−, Cd 2+ and Mn 2− is not explicable solely in terms of either inhibition of Ca-Mg-ATPase, or inhibition of Na-K-ATPase causing reduced intracellular K −. Our conclusions are similar to those previously obtained using synaptosomes and provide support for the hypothesis that inhibition of choline transport by Al 3+ may contribute to a number of disease states.

Apparent noncompetitive inhibition of choline transport in erythrocytes by inhibitors bound at the substrate site

According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zero trans entry and infinite trans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zero trans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zero trans and infinite trans exit was inhibited competitively, and zero trans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form.

Recovery of erythrocyte Li +/Na + countertransport and choline transport from lithium therapy

Li +/Na + countertransport, choline transport, and intracellular Li + and choline were measured in the RBC of a manic-depressive subject as a function of time after termination of Li + therapy. Countertransport recovered from its inhibition by Li + in 10 days. This recovery involved a decrease in the Michaelis-Menten parameter K m, without change in V max. RBC choline decreased, and choline influx rose, much more slowly back towards pre-Li + values over the course of two months. Thus, choline transport was still inhibited long after Li + levels in RBC and plasma had become undetectable. Measurements on age-separated RBC fractions showed that recovery of choline transport was mainly in the young-cell fraction. Hence inhibition of choline transport by Li + may be irreversible at the level of the individual RBC.

Choline transport in Saccharomyces cerevisiae.

Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated.