Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection.
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.
Barkhimer
Tatyana V
TV
Department of Chemistry, College of Arts & Sciences, University of Toledo, Toledo, OH 43606, USA.
Kirchhoff
Jon R
JR
Hudson
Richard A
RA
Messer
William S
WS
D000110:Acetylcholinesterase; D000429:Alcohol Oxidoreductases; D000818:Animals; D002396:Catechols / Q000494:pharmacology; D002794:Choline / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000493:pharmacokinetics*; D004563:Electrochemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004800:Enzymes, Immobilized; D007700:Kinetics; D008839:Microelectrodes; D010084:Oxidation-Reduction; D013574:Synaptosomes / Q000378:metabolism*
D002396:Catechols; D004800:Enzymes, Immobilized; C036022:3-trimethylaminomethylcatechol; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase; D002794:Choline