Inhibition Of High-affinity Choline Transport Research Articles

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy- N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.

Vitamin E attenuates the effects of both reversible and irreversible inhibitors of high-affinity choline transport in vivo

High-affinity choline transport (HAChT) is the rate limiting step in the synthesis of acetylcholine (ACh). The activity of HAChT and the binding of its selective inhibitor, [3H]hemicholinium-3 (HC-3) are affected by a number of exogenous and endogenous factors. Previous experiments demonstrated that Vitamin E pretreatment could prevent the decrease in HAChT and the cognitive deficits induced by the cholinotoxin AF64A [38]. To further examine this effect these experiments determined whether Vitamin E would alter the efficacy of both irreversible (AF64A) and reversible (HC-3) inhibitors of HAChT. In Experiment 1, rats were pretreated with Vitamin E (50 mg/kg), 24 h and 15 min, prior to bilateral icv injection of AF64A (0, 0.75, 1.5, or 3.0 nmol). HAChT was assessed in hippocampal synaptosomes, 14 days following surgery. Vitamin E prevented the dose-dependent AF64A-induced inhibition of HAChT in the hippocampus (HPC). In a second experiment, rats were pretreated with Vitamin E as above, and infused (icv) with the reversible inhibitor of HAChT, HC-3 (20 μg), or CSF. HAChT in the HPC was assessed 30 min, 4, 12, or 24 h after injection. HC-3 produced a significant decrease of HAChT (58%) that was maximal at 4 h and recovered by 24 h. Vitamin E significantly attenuated, but did not prevent, the inhibition of HAChT produced by HC-3. These experiments demonstrate that Vitamin E pretreatment can attenuate the effects of both reversible and irreversible inhibitors of HAChT. These data are discussed in terms of potential underlying mechanisms. It is possible that the neuroprotectant effects of Vitamin E on both reversible and irreversible inhibitors of HAChT reflect an action at the choline carrier and not an antioxidant effect.

Characterization of the irreversible inhibition of high-affinity choline transport produced by hemicholinium mustard.

The inhibition of high-affinity choline transport by hemicholinium mustard (HCM), an alkylating analogue of hemicholinium-3, was examined in rat brain synaptosomes and guinea pig myenteric plexus. In synaptosomes, 50% high-affinity choline transport inhibition occurs with an HCM concentration of 104 nM (4-min incubation). A 10-min preincubation with 10 microM HCM results in essentially complete (greater than 95%) inactivation that persists after washing. Low-affinity choline transport in synaptosomes is unaffected by HCM inhibition at all concentrations examined (1-50 microM). Time course experiments indicate that the maximum irreversible inhibition (58%) seen after a 1-min preincubation with 500 nM HCM decreases to 46% inhibition after a 15-min preincubation; however, analysis of variance reveals that this difference is not significant. HCM inhibition of acetylcholine release from myenteric plexus-longitudinal muscle preparations persists for at least 2 h after removal of drug from the incubation bath; this inactivation can be prevented by coincubation with a high choline concentration during treatment with the mustard. In contrast, inhibition produced by the parent compound hemicholinium-3 is largely reversed by washing in both preparations examined. The observed potency and selectivity of HCM suggest its usefulness as a covalent probe for high-affinity choline transport.

Hemicholinium-3 derivatives A-4 and A-5 affect choline and acetylcholine metabolism

The neuroblastoma-glioma hybrid cell (NG108-15) has a sodium-dependent, high-affinity choline transport system with a K m of 16.0 ± 3.4 μM and V max of 214.5 ± 27.7 pmol/min/mg protein. A-4, A-5 and HC-3 produce dose-dependent inhibition of high-affinity choline transport in NG108-15 cells. Following 24 h exposure to approximately the EC 50 of each inhibitor, no significant decrease was found in total choline accumulation or in choline incorporation into phosphatidylcholine. However, when additional inhibitor was added during the 24 h incubation, significant decreases in choline accumulation were produced by A-4 and A-5. Following 24 h exposure to each compound, only A-4 was able to significantly affect free choline content. In contrast, each inhibitor was able to significantly decrease acetylcholine content following 24 h exposure. Possible reasons for consistent decreases in acetylcholine versus minimal changes in choline metabolism will be discussed.

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.

Role of the aziridinium moiety in the in vivo cholinotoxicity of ethylcholine aziridinium ion (AF64A)

To assess the role of the aziridinium moiety for the cholinotoxicity of ethylcholine aziridinium ion (AF64A) we compared in vitro and in vivo effects of AF64A with those of various precursors as well as decomposition products of AF64A. In vitro, AF64A was the most effective irreversible inhibitor of high-affinity choline transport (HAChT) in hippocampal synaptosomes. The uncyclized precursor acetylethylcholine mustard and the acetylated form of AF64A were about 3 times less potent. Their potency, however, was reduced considerably when hydrolysis of the choline esters was prevented by physostigmine. Destruction of the aziridinium ring either by high pH (alcohol formation) or by thiosulfate (formation of Bunte salt) resulted in a loss of biological activity. This was also the case for the in vivo cholinotoxicity, as assessed by the decline in hippocampal concentration of acetylcholine (ACh) 7 days after intracerebroventricular (i.c.v.) infusion. The most pronounced reduction in ACh content was achieved after i.c.v. infusion of AF64A, whereas the precursor and the acetylated analog of AF64A induced a significant, but smaller reduction in the ACh content. These data indicate that the aziridinium ring of AF64A is essential for both the inhibition of HAChT in vitro and the cholinotoxicity in vivo. However, cyclization of the precursor compound as well as hydrolysis of acetylated AF64A also occur in tissue, leading to a partial activity of these compounds.

An evaluation of irreversible inhibition of synaptosomal high-affinity choline transport by choline mustard aziridinium ion.

Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC50 value for high-affinity transport was 0.94 microM, compared to 29 microM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 X 10(-2) s-1 for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.

Kinetic data on the inhibition of high-affinity choline transport into rat forebrain synaptosomes by choline-like compounds and nitrogen mustard analogues.

Abstract: A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high‐affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high‐affinity uptake (Ki=2.63, 3.15 and 2.72 μm, respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (Ki= 27.8 μm), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (Ki= 500 and 403 μm) as a blocker of choline transport. From study with these compounds it was concluded that the high‐affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10‐fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20‐fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high‐affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium‐3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high‐affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.

Inhibition of high-affinity choline transport in peripheral cholinergic endings by presynaptic snake venom neurotoxins.

USE of purified snake venom neurotoxins with potent curarimimetic action on peripheral cholinergic transmission (for example, α-bungarotoxin1) in the study of the biochemical properties of the nicotinic cholinoceptor has met with considerable success (see ref. 2 for review). Some of the crude venoms from which such toxins have been isolated also contain toxins which act presynaptically, such as β-bungarotoxin1, notexin3 and taipoxin4. These three toxins inhibit the release of acetylcholine by a mechanism which is independent of the release process per se3–7. We show here that in an in vitro membrane preparation8 derived from the cholinergic endings in Torpedo electric organ these and related toxins are potent inhibitors of the high-affinity choline transport system. These observations provide a biochemical explanation for the inhibitory action of these toxins on transmitter release as they effectively block re-synthesis of acetylcholine from choline.