Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection

Abstract

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.

Inhibition of choline transport by redox-active cholinomimetic bis-catechol reagents

“…Control experiments have shown two important points with respect to the CE-EC assay performance: 1) minimum Ch efflux was observed within the first 8 minutes of analysis indicating a negligible effect on the initial rate data collected during the time frame defined in the experimental section, and 2) non-specific transport was limited to at most 12% of the total transport observed. 19 The dose-response curve also offers the convenience of comparing the relative inhibitor potencies of multiple inhibitors for CHT provided the Ch substrate concentration remains constant for all experiments. 25 for our experiments, the Ch concentration was fixed at 2 μM for ease of comparison to the monocatechol reagents.…”

“…Ch concentrations were analyzed using BuCh as an internal standard. 17,18 Evaluation of the inhibition properties of 6 and 7 utilized the Ch transport assay methods developed by Barkhimer et al 11,19 using mouse synaptosomes as the CHT model. Synaptosome suspensions were prepared from C57BL6 adult male mice (Harlan Sprague Dawley, Indianapolis, IN) following the general procedure of Gray and Whittaker, 23 as modified by Patel.…”

Synthesis and characterization of luminescent cadmium selenide/zinc selenide/zinc sulfide cholinomimetic quantum dots

“…In our laboratory, analyses of ACh and Ch were achieved in vitro and in vivo by use of capillary electrophoresis with electrochemical detection where sample sizes of 5-10 nL and attomole detection limits were possible. [14][15][16][17][18][19][20] These methods were also valuable to monitor small timedependent changes in Ch concentrations in near real-time during the transport of Ch through CHT. Determination of K I and IC 50 values, and the mode of inhibition for inhibitors of CHT were then possible.…”

Electrocatalytic Microelectrode Detectors for Choline and Acetylcholine Following Separation by Capillary Electrophoresis

“…Detection limits of 100 amol for Ch and 1 fmol for ACh were possible using this electrode as the detector for CE coupled with an internal standard method 8,16. This approach was subsequently used for evaluation of Ch transport rates in the presence of a new class of quaternary ammonium alkyl-substituted inhibiters of CHT 17–19. By varying the efficacy of the inhibitor, differences in Ch transport rates and quantitative evaluation of their potency were determined.…”

“…8,16 This approach was subsequently used for evaluation of Ch transport rates in the presence of a new class of quaternary ammonium alkyl-substituted inhibiters of CHT. [17][18][19] By varying the efficacy of the inhibitor, differences in Ch transport rates and quantitative evaluation of their potency were determined. An alternative detector design was developed for this purpose by covalent attachment of the enzymes to a platinum electrode.…”