AF64A-induced cytotoxicity and changes in choline acetyltransferase activity in the LA-N-2 neuroblastoma cell line are modulated by choline and hemicholinium-3.
The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 microM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10(-5), 10(-4) and 10(-3) M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10(-3) M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.
Santiago
L R
LR
Department of Pharmacology, Loyola University at Chicago, Stritch School of Medicine, Maywood, IL 60153, USA.
Ivy
M T
MT
Erickson
L C
LC
Hanin
I
I
D000818:Animals; D001388:Aziridines / Q000494:pharmacology*; D002794:Choline / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006426:Hemicholinium 3 / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D009466:Neuromuscular Blocking Agents / Q000494:pharmacology*; D051381:Rats
D001388:Aziridines; D009466:Neuromuscular Blocking Agents; D006426:Hemicholinium 3; C044894:ethylcholine aziridinium; D002795:Choline O-Acetyltransferase; D002794:Choline