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.     

Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium

Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 m. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 m. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min^–1. The rate constant of ECMA attack on the carrier is about 440m ^–1 sec^–1.     

Dose- and time-dependent hippocampal cholinergic lesions induced by ethylcholine mustard aziridinium ion: Effects of nerve growth factor,GM1 ganglioside,and vitamin E

Ethylcholine mustard aziridinium ion (ECMA) was infused intracerebroventricularly (icv) to rats followed by measurement of two markers of presynaptic cholinergic neurons, choline acetyltransferase (ChAT) activity and high affinity choline transport (HAChT), in the hippocampus and cortex. Bilateral icv administration of 1, 2, or 3 nmol of ECMA per side produced dose-dependent reductions in each marker in the hippocampus, but not in the cortex, one week after treatment. Reductions of 52% and 46% for ChAT activity and HAChT, respectively, were produced in the hippocampus by 3 nmol ECMA. Measurement of these two markers at different times after icv infusion of 2 nmol ECMA/ventricle revealed that the activity of ChAT was reduced to a greater extent than was HAChT in the hippocampus 1 day and 1, 2, 4, and 6 weeks after treatment. The maximal reductions of ChAT activity and HAChT (61% and 53%, respectively) were reached between 1 and 2 weeks after ECMA administration. There was no evidence of regeneration of either marker at 4 or 6 weeks posttreatment. HAChT and ChAT activity in the cortex were not altered at any of the posttreatment times examined.ECMA-induced deficits in hippocampal ChAT activity and HAChT were not counteracted by the following treatments: (i) daily administration of GM₁ ganglioside (10 mg/kg, intraperitoneally (ip)) from the day prior to infusion of ECMA until 2 weeks later; (ii) daily administration of GM₁ ganglioside between 2 and 6 weeks after infusion of ECMA; and (iii) icv administration of nerve growth factor (NGF) twice per week for 2 weeks after ECMA treatment. Since similar treatments with NGF and GM₁ ganglioside ameliorate lesions induced by other methods, these results indicate that the mechanism of lesion formation and the surviving cellular components influence the functional effects of neurotrophic factors. In contrast to the above results, treatment with vitamin E significantly attenuated ECMA-induced deficits of ChAT activity and HAChT. Further studies of the effects of vitamin E on the development of ECMA-induced deficits may help to elucidate the mechanism action of ECMA.     

A comparative study of high affinity choline uptake and choline utilization in cholinergic and non-cholinergic tissues

Comparative studies of [3H]choline accumulation were done in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion. Dual uptake processes for choline were found in all three tissues. In acute experiments, the corpora pedunculata high affinity choline uptake system showed exclusive sensitivity to ouabain. Prolonged exposure to ouabain revealed that the HAChUS of all three tissues were significantly inhibited. The metabolism of [3H]choline transported via the high affinity process in the three tissues was studied. [3H]Acetylcholine was a major product of the [3H]choline taken up by the corpora pedunculata and the abdominal ganglia. Phosphorylcholine was the major product seen in cardiac ganglion extracts and occurred in significant proportions in abdominal ganglia extracts. [3H]Acetylcholine was not detected in cardiac ganglion extracts. Treatment with either lithium chloride or hemicholinium-3 markedly inhibited high affinity uptake of [3H]choline in all three tissues.     

Carrier-mediated inhibition of choline acetyltransferase

Incubation of rat forebrain synaptosomes with choline mustard aziridinium ion in a sodium-rich medium caused a time-dependent inhibition of the high-affinity transport of choline, as well as a significant decrease in intrasynaptosomal choline acetyltransferase activity. In the absence of added sodium choline uptake by a sodium-independent mechanism was also blocked in a time-dependent manner but intrasynaptosomal choline acetyl-transferase activity was unaltered. Neither monoethylcholine nor hemicholinium-3 changed intrasynaptosomal choline acetyl-transferase activity but competitively inhibited the transport of choline. The results indicate that there may be a fraction of choline acetyltransferase that is closely associated with the sodium-dependent high-affinity choline transport system and that this fraction can be irreversibly inhibited by choline mustard aziridinium ion, perhaps indirectly mediated by alkylation of the carrier.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

Stereospecificity of High-and Low-Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.     

The effect of hemicholinium-3 on choline and acetylcholine levels in a sympathetic ganglion.

Pregangliaaonic stimulation of the cat's superior cervical ganglion in the presence of hemicholinium-3 (HC-3) produced the expected depletion of acetylcholine (ACh) stores, but failed to cause a corresponding reduction in the choline content. These results suggest that either HC-3 possesses an intracellular site of action or that in lower doses it selectively inhibits a specialized choline transport system in cholinergic nerves. At a dose of 2 mg/kg, HC-3 probably blocked ACh synthesis completely in ganglia stimulated at 20 Hz. Under these conditions, there was a rapid depletion of ACh to about 50% of control levels during the first 5 min of stimulation and thereafter the rate of decline in ACh levels proceeded at a much slower pace. Since the 2 mg/kg dose of HC-3 did not raise plasma choline concentrations, it may be assumed that non-specialized choline transport systems in other tissues were not significantly inhibited by this dose of HC-3. However, when the dose of HC-3 was increased to 4 mg/kg, plasma choline levels increased by 58%.     

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.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

HIGH AFFINITY CHOLINE TRANSPORT IN GUINEA PIG BRAIN AND THE EFFECT OF NOREPINEPHRINE

—The influence of 1-norepinephrine on the accumulation of [¹⁴C]choline by nuclei-free homogenates and synaptosomes of guinea-pig brain was studied. Kinetic analysis of choline accumulation by guinea-pig brain resulted in both high and low affinity Michaelis constants. Norepinephrine stimulated the high affinity choline transport process but not the low and the magnitude of its stimulation in 3 different brain regions was correlated with the choline acetyltransferase activity of those regions. Depletion of norepinephrine from the brainstem by pretreatment with the catecholamine depleter alpha-methyl-para-tyrosine significantly decreased the maximal velocity of choline transport. Both the alpha adrenergic receptor blocker phentolamine and the beta adrenergic receptor blocker propranalol inhibited norepinephrine induced stimulation of choline transport. Cocaine stimulated choline transport at low concentrations and pretreatment of animals with reserpine significantly antagonized cocaine's stimulation of choline transport. The results suggest that endogenous norepinephrine may modify the high affinity choline transport process in guinea-pig brain.     

Reconstitution of carrier-mediated choline transport in proteoliposomes prepared from presynaptic membranes oftorpedo electric organ,and its internal and external ionic requirements

Summary Proteoliposomes made by a butanol-sonication technique from electric organ presynaptic membranes showed choline transport activity. In contrast to intact nerve terminals, the uptake of choline was dissociated from its conversion to acetylcholine in this preparation. The kinetics of choline uptake by proteoliposomes was best described by two Michaelis-Menten components. At a low concentration of choline, uptake was inhibited by hemicholinium-3 and required external Na⁺ and, thus, closely resembled high-affinity choline uptake by intact cholinergic nerve terminals. Choline transport could be driven by the Na⁺ gradient and by the transmembrane potential (inside negative) but did not directly require ATP. External Cl^–, but not a Cl^– gradient, was needed for choline transport activity. It is suggested that internal K⁺ plays a role in the retention of choline inside the proteoliposome. Proteoliposomes should prove a useful tool for both biochemical and functional studies of the highaffinity choline carrier.Abbreviations ACh acetylcholine - HC-3 hemicholinium-3 - ChAT choline acetyltransferase     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes.

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K+ and extrude Na+ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na+, Li+, Rb+, Cs+ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na+ gradient gave a maximal value of about 10(2). A secondary active transport of choline, where Na+ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.     

Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na(+) from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na(+)-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H(+) gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.     

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na⁺ from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na⁺-free conditions was inhibited by a Na⁺/H⁺ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

Studies on the characterization of rat prostate androgen receptors

Summary Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km=3 µM) and another of low affinity (Km=400 µM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na⁺ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.