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.