AF64A-INDUCED CYTOTOXICITY AND CHANGES IN CHOLINE-ACETYLTRANSFERASE ACTIVITY IN THE LA-N-2 NEUROBLASTOMA CELL-LINE ARE MODULATED BY CHOLINEAND HEMICHOLINIUM-3

The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been u sed to selectively destroy the cholinergic system. Due to its structur al similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAC hT) system to produce persistent and selective cholinergic deficits. T he mechanism by which it exerts its cholinotoxicity remains to be eluc idated. We have examined the effects of AF64A in the human neuroblasto ma 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 mu M) produced dose-dependent increases in cell kill as m easured by colony formation assay. The addition of increasing concentr ations (10(-5), 10(-4) and 10(-3) M) of choline and hemicholinium-3 (H C-3) protected the cells from the cytotoxic effects of AF64A. At the s ame doses, AF64A also decreased choline acetyltransferase (ChAT) activ ity. In the presence of the highest concentration of choline or KC-3 ( 10(-3) M) which produced complete protection against AF64A's cytotoxic ity in the colony formation assay, ChAT activity was restored to contr ol values. These results demonstrate that agents that utilize (i.e., c holine) or inhibit (i.e., HC-3) the choline uptake system prevented AF 64A-induced cytotoxicity and decreases in ChAT activity, in a manner s imilar to that which has been observed in chick and rat primary cholin ergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serve s well as an in vitro model of the cholinergic neuron and provides a u seful system to study the mode of cholinotoxicity induced by AF64A.