The
choline analogue
ethylcholine mustard aziridinium (
AF64A) is a potent and irreversible inhibitor of
choline uptake in brain synaptosomes and is used as a
neurotoxin to produce animal models of
cholinergic hypofunction. However, previous studies have shown that intraocular administration of
AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether
AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of
lactate dehydrogenase activity in the culture medium,
AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human
cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of
AF64A correlated well with the affinity of the
choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell
choline carrier, as demonstrated by the following observations: (a)
AF64A inhibited [3H]
choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of
choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.