Inhibition Of Choline Transport Research Articles

Effects of acetylcholine mustard aziridinium ion and its choline analogue on choline transport into synaptosomes.

A compound thought to be choline mustard aziridimium ion was found previously in our laboratory to inhibit choline transport into human erythrocytes. Experiments with synaptosomes prepared from brain of rat now show that the aziridinium ion of choline mustard hydrochloride inhibited high- and low-affinity choline transport, with the inhibitory effect being about 10 times less than that of hemicholinium-3. Acetylcholine mustard aziridinium ion also inhibited the transport of choline, but it was less potent than its choline analogue. Studies with choline mustard aziridinium ion may yield important information about the transport of substances into the presynaptic nerve terminal.

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.

Membrane effects of cytochalasin B. Competitive inhibition of facilitated diffusion processes in rat hepatoma cells and other cell lines and effect on formation of functional transport sites.

Cytochalasin B competitively inhibits the transport of 2-deoxy-D-glucose and thymidine in a number of different cell lines (Novikoff rat hepatoma cells, mouse L, S180 and Ki-MSV-transformed BALB/3T3 cells, and human HeLa cells). The apparent Km values for the transport of these substrates as well as the apparent Ki values for the inhibition by cytochalasin B are very similar for the various cell lines, and the effect is readily and completely reversed by removal of the chemical. Thymidine transport by Chinese hamster ovary cells however, is little affected by cytochalasin B, whereas the transport of 2-deoxy-D-glucose, uridine and guanine by these cells is competitively inhibited to about the same extent as in other cell lines. In addition and concomitant with the inhibition of cytokinesis and an alteration in cell shape, cytochalasin B also impairs and delays the formation of functional transport sites for thymidine, guanine and choline in synchronized populations of Novikoff cells without affecting the apparent affinities of the transport systems for their substrates. This effect is unrelated to the direct inhibition of the transport processes, since the drug does not directly inhibit choline transport and has no effect on the formation of 2-deoxy-D-glucose transport sites in spite of the fact that it strongly inhibits the transport of this substrate. The inhibition of functional transport sites may be due to the induction of a structural alteration in the membrane by cytochalasin B which impairs the insertion of new proteins of certain but not all transport systems into the membrane.

LITHIUM TREATMENT STRONGLY INHIBITS CHOLINE TRANSPORT IN HUMAN ERYTHROCYTES

1 The influx of [(14)C]-choline and the efflux of (22)Na in human erythrocytes were measured in vitro using blood from patients treated with lithium, patients not on lithium and healthy individuals. 2 The administration of lithium to patients significantly reduces the transport of choline; during the first 6 weeks of treatment the influx of choline is about half the normal rate, later it falls to around 10%. 3 This inhibition of choline transport is not dependent on the presence of lithium in the incubation medium. 4 The active and passive efflux of sodium are apparently not affected by lithium treatment.

Some properties of an SH group essential for choline transport in human erythrocytes.

1. The choline transport system in human erythrocytes is inhibited by N-ethylmaleimide (NEM), cystamine and p-chloromercuribenzene sulphonic acid (PCMBS).2. External choline increases the rate of inhibition by NEM and cystamine but decreases the rate of inhibition by PCMBS. Intracellular choline has the opposite effect.3. Competitive inhibitors of choline transport that are not themselves transported protect the carrier against all three thiol reagents.4. Some thiol reagents with a very low lipid solubility do not inhibit choline transport.5. The transport inhibition by cystamine is reversed by various reducing agents.6. Cystamine protects the transport system against NEM and PCMBS.7. It is suggested that NEM and cystamine react with an SH group of the transport system and that this SH group is more reactive when the carrier is facing inside. PCMBS penetrates erythrocytes only very slowly and is assumed to react preferentially with the outward facing carrier.8. The reactive SH group seems to be located in a lipophilic environment.

Effects of quaternary ammonium compounds on choline transport in red cells.

1. Neuromuscular blocking agents have been shown to be powerful inhibitors of choline transport in human erythrocytes. Ganglionic blocking agents were weaker inhibitors.2. The affinity of the choline transport site for alkyltrimethylammonium compounds was considerably higher than its affinity for alkyl-bis-(trimethylammonium) compounds of similar chain length. The affinity increases with increasing length of the alkyl chain in both series.3. Tetramethylammonium (TMA), ethyl- and propyltrimethylammonium appear to enter the cells on the choline carrier while the larger monoquaternary compounds, and the bisquaternary compounds, bind to the carrier but are unable to cross the cell membrane.4. Radioactively labelled carbachol, acetylcholine and decamethonium do not enter the cells on the choline carrier.5. Choline transport in erythrocytes from patients with myasthenia gravis is normal, suggesting that this disorder is not associated with a generalized defect of choline transport.